Pesticidal genes and methods of use

ABSTRACT

Compositions having pesticidal activity and methods for their use are provided. Compositions include isolated and recombinant polypeptide sequences having pesticidal activity, recombinant and synthetic nucleic acid molecules encoding the pesticidal polypeptides, DNA constructs comprising the nucleic acid molecules, vectors comprising the nucleic acid molecules, host cells comprising the vectors, and antibodies to the pesticidal polypeptides. Nucleotide sequences encoding the polypeptides provided herein can be used in DNA constructs or expression cassettes for transformation and expression in organisms of interest, including microorganisms and plants. The compositions and methods provided herein are useful for the production of organisms with enhanced pest resistance or tolerance. Transgenic plants and seeds comprising a nucleotide sequence that encodes a pesticidal protein of the invention are also provided. Such plants are resistant to insects and other pests. Methods are provided for producing the various polypeptides disclosed herein, and for using those polypeptides for controlling or killing a pest. Methods and kits for detecting polypeptides of the invention in a sample are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/948,534, filed Apr. 9, 2018, which claims priority from U.S.Provisional Application Nos. 62/543,543, filed Aug. 10, 2017, and62/484,107, filed Apr. 11, 2017, which applications are herebyincorporated in their entirety by referenced in this application.

FIELD OF THE INVENTION

The invention is drawn to methods and compositions for controllingpests, particularly plant pests.

BACKGROUND

Pests, plant diseases, and weeds can be serious threats to crops. Lossesdue to pests and diseases have been estimated at 37% of the agriculturalproduction worldwide, with 13% due to insects, bacteria and otherorganisms.

Toxins are virulence determinants that play an important role inmicrobial pathogenicity and/or evasion of the host immune response.Toxins from the gram-positive bacterium Bacillus, particularly Bacillusthuringensis, have been used as insecticidal proteins. Currentstrategies use the genes expressing these toxins to produce transgeniccrops. Transgenic crops expressing insecticidal protein toxins are usedto combat crop damage from insects.

While the use of Bacillus toxins has been successful in controllinginsects, resistance to Bt toxins has developed in some target pests inmany parts of the world where such toxins have been used intensively.One way of solving this problem is sowing Bt crops with alternating rowsof regular non Bt crops (refuge). An alternative method to avoid or slowdown development of insect resistance is stacking insecticidal geneswith different modes of action against insects in transgenic plants. Thecurrent strategy of using transgenic crops expressing insecticidalprotein toxins is placing increasing emphasis on the discovery of noveltoxins, beyond those already derived from the bacterium Bacillusthuringiensis. These toxins may prove useful as alternatives to thosederived from B. thuringiensis for deployment in insect- andpest-resistant transgenic plants. Thus, new toxin proteins are needed.

SUMMARY

Compositions having pesticidal activity and methods for their use areprovided. Compositions include isolated and recombinant polypeptidesequences having pesticidal activity, recombinant and synthetic nucleicacid molecules encoding the pesticidal polypeptides, DNA constructscomprising the nucleic acid molecules, vectors comprising the nucleicacid molecules, host cells comprising the vectors, and antibodies to thepesticidal polypeptides. Nucleotide sequences encoding the polypeptidesprovided herein can be used in DNA constructs or expression cassettesfor transformation and expression in organisms of interest, includingmicroorganisms and plants.

The compositions and methods provided herein are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. Transgenic plants and seeds comprising anucleotide sequence that encodes a pesticidal protein of the inventionare also provided. Such plants are resistant to insects and other pests.

Methods are provided for producing the various polypeptides disclosedherein, and for using those polypeptides for controlling or killing apest. Methods and kits for detecting polypeptides of the invention in asample are also included.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

I. Polynucleotides and Polypeptides

Compositions and method for conferring pesticidal activity to anorganism are provided. The modified organism exhibits pesticidalresistance or tolerance. Recombinant pesticidal proteins, orpolypeptides and fragments and variants thereof that retain pesticidalactivity, are provided and include those set forth in SEQ ID NOs: 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and/or 205. Thepesticidal proteins are biologically active (e.g., pesticidal) againstpests including insects, fungi, nematodes, and the like. Nucleotidesencoding the pesticidal polypeptides, including for example, SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and/or 205or active fragments or variants thereof, can be used to producetransgenic organisms, such as plants and microorganisms. The pesticidalproteins are biologically active (for example, are pesticidal) againstpests including insects, fungi, nematodes, and the like. In specificembodiments, the pesticidal polypeptides and the active variant andfragments thereof have an improved pesticidal activity when compared toother polypeptides in the art. Polynucleotides encoding the pesticidalpolypeptides, including for example, SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, and/or 205 or active fragmentsor variants thereof, can be used to produce transgenic organisms, suchas plants and microorganisms. The transformed organisms arecharacterized by genomes that comprise at least one stably incorporatedDNA construct comprising a coding sequence for a pesticidal proteindisclosed herein. In some embodiments, the coding sequence is operablylinked to a promoter that drives expression of the encoded pesticidalpolypeptide. Accordingly, transformed microorganisms, plant cells, planttissues, plants, seeds, and plant parts are provided. A summary ofvarious polypeptides, active variants and fragments thereof, andpolynucleotides encoding the same are set forth below in Table 1. Asnoted in Table 1, various forms of polypeptides are provided. Fulllength pesticidal polypeptides, as well as, modified versions of theoriginal full-length sequence (i.e., variants) are provided. Table 1further denotes “CryBP1” sequences. Such sequences comprise accessorypolypeptides that can be associated with some of the toxin genes. Insuch instances, the CryBP1 sequences can be used alone or in combinationwith any of the pesticidal polypeptides provided herein. Table 1 furtherprovides Split-Cry C-terminus polypeptides. Such sequence comprise thesequence of a downstream protein that has homology to the C-terminal endof the Cry class of toxin genes and are usually found after a Cry genethat is not full-length and is missing the expected C-terminal region.

TABLE 1 Summary of SEQ ID NOs, Gene Class, and Variants thereof- Split-Modi- CryB Cry C- fied P1 terminus Seq Seq Seq Seq Gene APG ID ID ID(s)ID ID Class % Identity % Similarity Homologs APG00837.0 1 Bin 65, 70,75, 80, 85, 90, 75, 80, 85, 90, 91, WP_002090518.1 (72.48% identity,83.65% 91, 92, 93, 94, 95, 96, 92, 93, 94, 95, 96, similarity) 97, 98,99, 100 97, 98, 99, 100 WP_048517129.1 (72.48% identity, 83.65%similarity) WP_002167240.1 (62.15% identity, 74.03% similarity)APG00386.0 US_2016_0311864_A1-158 (60.33% identity, 72.18% similarity)WP_016093722.1 (55.1% identity, 71.07% similarity) APG00880.0US_2016_0366881_A1-134 (53.28% identity, 65.57% similarity) APG00215.0US_2016_0311864_A1-78 (53.02% identity, 68.68% similarity) APG06051.0(50.69% identity, 63.91% similarity) WP_042162506.1 (41.98% identity,59.09% similarity) APG03147.0 (40.75% identity, 55.5% similarity)WP_002016877.1 (40.69% identity, 57.18% similarity) APG01013.0 2  3 Cry50, 55, 60, 65, 70, 75, 65, 70, 75, 80, 85, WP_002169796.1 (73.77%identity, 83.25% 80, 85, 90, 91, 92, 93, 90, 91, 92, 93, 94, similarity)94, 95, 96, 97, 98, 99, 95, 96, 97, 98, 99, US_8461421_B2-264 (49.14%identity, 63.52% 100 100 similarity) US_8461421_B2-140 (47.76% identity,61.74% similarity) APG08350.0 (47.23% identity, 60.14% similarity)APG00044.0 US_2016_0304898_A1-71 (46.53% identity, 62.61% similarity)CA_2866166-375 (45.43% identity, 59.6% similarity) CA_2866166-378(45.37% identity, 61.28% similarity) APG00041.0 US_2016_0304898_A1-64(42.24% identity, 57.88% similarity) CA_2866166-376 (40.34% identity,55.75% similarity) AEH76822.1 (39.93% identity, 55.24% similarity)US_8461421_B2-84 (39.7% identity, 54.92% similarity) CA_2844913-189(39.55% identity, 45.4% similarity) CA_2844913-190 (39.55% identity,45.27% similarity) WO_2015_118207-1 (39.02% identity, 54.86% similarity)APG01199.0 (38.02% identity, 50.18% similarity) WP_002187593.1 (37.92%identity, 52.63% similarity) APG00145.0 US_2016_0304898_A1-163 (36.66%identity, 53.7% similarity) APG00140.0 US_2016_0177333_A1-78 (36.66%identity, 53.0% similarity) APG00534.0 (36.54% identity, 51.38%similarity) APG06630.0 (36.04% identity, 49.16% similarity) APG00897.0US_2016_0355842_A1-186 (35.92% identity, 48.81% similarity)CA_2844913-180 (35.23% identity, 38.57% similarity) APG01155.0 4  5 MTX90, 91, 92, 93, 94, 95, 94, 95, 96, 97, 98, APG08085.0 (89.94% identity,93.83% similarity) 96, 97, 98, 99, 100 99, 100 WP_044444098.1 (88.6%identity, 93.49% similarity) APG00743.0 (87.95% identity, 93.49%similarity) APG02555.0 (87.66% identity, 92.53% similarity) APG02782.0(84.76% identity, 88.72% similarity) APG02960.0 (83.13% identity, 88.04%similarity) SFC26517.1 (82.08% identity, 89.25% similarity) APG04643.0(82.01% identity, 87.5% similarity) WP_060749709.1 (81.82% identity,90.91% similarity) APG04485.0 (56.11% identity, 70.53% similarity)APG03368.0 (55.56% identity, 70.16% similarity) APG01705.0 (55.24%identity, 70.16% similarity) APG01189.0 6  7 MTX 94, 95, 96, 97, 98, 99,97, 98, 99, 100 APG00187.0 US_2016_0355842_A1-20 (93.73% 100 identity,96.12% similarity) APG00218.0 US_2016_0355842_A1-26 (57.18% identity,69.21% similarity) APG00245.0 US_2016_0355842_A1-34 (56.89% identity,68.62% similarity) APG03900.0 (56.23% identity, 69.57% similarity)US_8829279_B2-42 (28.7% identity, 44.93% similarity) APG01363.0 8  9 Bin90, 91, 92, 93, 94, 95, 93, 94, 95, 96, 97, APG00229.0US_2016_0311864_A1-84 (89.36% 96, 97, 98, 99, 100 98, 99, 100 identity,92.08% similarity) APG07334.0 (88.86% identity, 92.66% similarity)APG00419.0 US_2016_0311864_A1-176 (87.97% identity, 91.23% similarity)APG00353.0 US_2016_0311864_A1-144 (84.81% identity, 89.11% similarity)APG00301.0 US_2016_0311864_A1-131 (84.56% identity, 88.35% similarity)APG00844.0 US_2016_0311864_A1-221 (84.3% identity, 87.59% similarity)APG00203.0 US_2016_0311864_A1-69 (83.54% identity, 88.35% similarity)APG00412.0 US_2016_0311864_A1-173 (80.2% identity, 85.4% similarity)APG00300.0 US_2016_0355842_A1-53 (78.75% identity, 87.5% similarity)APG00065.0 US_2016_0177333_A1-31 (78.27% identity, 86.42% similarity)APG09977.0 (76.77% identity, 85.1% similarity) APG00724.0 (76.67%identity, 83.37% similarity) APG01502.0 10  11 Cyt 90, 91, 92, 93, 94,95, 92, 93, 94, 95, 96, APG04725.0 (85.96% identity, 91.06% similarity)96, 97, 98, 99, 100 97, 98, 99, 100 APG00126.0 US_2016_0304898_A1-153(83.4% identity, 91.7% similarity) WP_016110460.1 (82.64% identity,89.67% similarity) APG08631.0 (78.49% identity, 86.85% similarity)APG00437.0 US_2016_0366881_A1-62 (77.29% identity, 84.06% similarity)APG07961.0 (56.56% identity, 72.13% similarity) APG00128.0US_2016_0304898_A1-157 (54.44% identity, 69.35% similarity) APG08230.0(54.18% identity, 71.31% similarity) WP_016110459.1 (53.63% identity,68.55% similarity) APG02923.0 (53.01% identity, 69.08% similarity)APG00177.0 US_2016_0311864_A1-52 (52.71% identity, 68.99% similarity)APG00121.0 US_2016_0311864_A1-29 (51.82% identity, 65.33% similarity)APG01844.0 12  13 14 Cry 55, 60, 65, 70, 75, 80, 65, 70, 75, 80, 85,CA_2866166-428 (53.25% identity, 63.84% 85, 90, 91, 92, 93, 94, 90, 91,92, 93, 94, similarity) 95, 96, 97, 98, 99, 100 95, 96, 97, 98, 99,APG03786.0 (40.36% identity, 54.09% similarity) 100 APG04671.0 (39.12%identity, 55.37% similarity) CA_2866166-242 (37.82% identity, 56.02%similarity) AEX56526.1 (37.6% identity, 53.58% similarity) CA_2516349-47(36.88% identity, 53.18% similarity) WP_044875982.1 (36.02% identity,51.41% similarity) APG00204.0 US_2016_0177333_A1-89 (35.95% identity,53.07% similarity) AHL90402.1 (35.58% identity, 51.37% similarity)CA_2866166-243 (35.58% identity, 51.24% similarity) CA_2866166-374(35.39% identity, 52.44% similarity) APG00695.0 (35.09% identity, 51.36%similarity) APG01927.0 15 Vip4 70, 75, 80, 85, 90, 91, 80, 85, 90, 91,92, SCC34829.1 (67.51% identity, 77.95% similarity) 92, 93, 94, 95, 96,97, 93, 94, 95, 96, 97, US_2016_0339078_A1-28464 (66.77% identity, 98,99, 100 98, 99, 100 76.82% similarity) APG00106.0 US_2016_0304898_A1-134(64.39% identity, 73.35% similarity) APG09984.0 (61.08% identity, 71.85%similarity) AGT29561.1 (55.96% identity, 68.57% similarity) APG04069.0(50.75% identity, 64.49% similarity) APG01474.0 (50.69% identity, 65.19%similarity) US_2016_0339078_A1-29674 (49.8% identity, 65.46% similarity)WP_016123960.1 (49.8% identity, 63.67% similarity) APG07526.0 (48.59%identity, 62.59% similarity) CA_2844913-129 (47.82% identity, 60.12%similarity) CA_2844913-130 (47.82% identity, 60.12% similarity)APG04931.0 (47.52% identity, 61.22% similarity) SCC38903.1 (46.92%identity, 62.56% similarity) WP_053512697.1 (46.75% identity, 61.49%similarity) US_2016_0339078_A1-28124 (46.6% identity, 59.9% similarity)SCL96146.1 (46.56% identity, 61.3% similarity) WP_048561796.1 (46.46%identity, 61.49% similarity) APG07790.0 (45.55% identity, 58.99%similarity) APG01969.0 16  17 MTX 90, 91, 92, 93, 94, 95, 94, 95, 96,97, 98, APG00384.0 US_2016_0366881_A1-44 (86.05% 96, 97, 98, 99, 100 99,100 identity, 93.02% similarity) APG03015.0 (57.67% identity, 70.74%similarity) APG00344.0 US_2016_0366881_A1-22 (56.66% identity, 71.1%similarity) APG00477.0 US_2016_0355842_A1-99 (54.86% identity, 69.71%similarity) WP_016098287.1 (54.73% identity, 69.91% similarity)WP_016099611.1 (53.56% identity, 68.09% similarity) WP_016098181.1(48.6% identity, 62.85% similarity) APG00325.0 US_2016_0366881_A1-12(47.91% identity, 64.62% similarity) APG02060.0 (47.75% identity, 63.48%similarity) APG00092.0 US_2016_0304898_A1-122 (45.74% identity, 65.06%similarity) APG00982.0 US_2016_0304898_A1-226 (45.17% identity, 64.49%similarity) APG01971.0 18  19 Cyt 75, 80, 85, 90, 91, 92, 85, 90, 91,92, 93, APG00017.0 US_2016_0304898_A1-28 (71.3% 93, 94, 95, 96, 97, 98,94, 95, 96, 97, 98, identity, 82.06% similarity) 99, 100 99, 100APG01309.0 (69.96% identity, 81.61% similarity) APG00464.0US_2016_0366881_A1-78 (68.61% identity, 81.17% similarity) APG00194.0US_2016_0311864_A1-65 (67.86% identity, 81.7% similarity)US_9121035_B2-20 (33.33% identity, 53.33% similarity) US_9121035_B2-6(33.33% identity, 53.33% similarity) US_8513493_B2-47 (32.5% identity,52.5% similarity) US_9121035_B2-4 (32.5% identity, 52.5% similarity)US_9121035_B2-15 (31.97% identity, 51.64% similarity) 2RCI_A (26.07%identity, 43.16% similarity) APG02013.0 20  21 MTX 65, 70, 75, 80, 85,90, 70, 75, 80, 85, 90, WP_000794513.1 (62.2% identity, 68.45% 91, 92,93, 94, 95, 96, 91, 92, 93, 94, 95, similarity) 97, 98, 99, 100 96, 97,98, 99, 100 US_8461421_B2-117 (61.65% identity, 67.85% similarity)APG00254.0 US_2016_0355842_A1-40 (37.55% identity, 51.99% similarity)WP_061885189.1 (37.5% identity, 51.49% similarity) APG08369.0 (36.53%identity, 48.5% similarity) WP_046655236.1 (36.46% identity, 48.96%similarity) ETK27180.1 (36.25% identity, 49.38% similarity)WP_006284936.1 (36.23% identity, 48.5% similarity) APG00541.0US_2016_0355842_A1-119 (36.04% identity, 47.15% similarity) APG00244.0US_2016_0355842_A1-33 (36.04% identity, 46.85% similarity) APG00509.0US_2016_0355842_A1-113 (35.74% identity, 46.85% similarity) APG00552.0US_2016_0366881_A1-110 (35.69% identity, 48.08% similarity)WP_051427903.1 (35.42% identity, 46.53% similarity) WP_036654376.1(35.26% identity, 48.02% similarity) APG02060.0 22  23 MTX 50, 55, 60,65, 70, 75, 70, 75, 80, 85, 90, WP_016098181.1 (83.97% identity, 89.8%80, 85, 90, 91, 92, 93, 91, 92, 93, 94, 95, similarity) 94, 95, 96, 97,98, 99, 96, 97, 98, 99, 100 APG00344.0 US_2016_0366881_A1-22 (48.04% 100identity, 66.2% similarity) APG00384.0 US_2016_0366881_A1-44 (47.74%identity, 63.56% similarity) APG01969.0 (47.62% identity, 63.31%similarity) APG03015.0 (47.09% identity, 63.99% similarity) APG00477.0US_2016_0355842_A1-99 (46.7% identity, 61.54% similarity) WP_016098287.1(45.96% identity, 63.23% similarity) APG00092.0 US_2016_0304898_A1-122(43.79% identity, 61.86% similarity) APG00982.0 US_2016_0304898_A1-226(43.5% identity, 62.43% similarity) WP_016099611.1 (38.59% identity,55.21% similarity) APG00325.0 US_2016_0366881_A1-12 (37.81% identity,54.52% similarity) APG02123.0 24  25 MTX 80, 85, 90, 91, 92, 93, 90, 91,92, 93, 94, WP_002193657.1 (81.96% identity, 90.51% 94, 95, 96, 97, 98,99, 95, 96, 97, 98, 99, similarity) 100 100 WP_033671297.1 (81.79%identity, 91.37% similarity) EOQ05487.1 (80.19% identity, 89.94%similarity) WP_006097189.1 (80.19% identity, 89.14% similarity)WP_040119245.1 (79.87% identity, 89.14% similarity) WP_040118692.1(79.55% identity, 88.82% similarity) WP_018783466.1 (76.75% identity,87.58% similarity) APG06989.0 (75.4% identity, 87.86% similarity)APG05651.0 (75.4% identity, 85.62% similarity) WP_016131662.1 (75.4%identity, 85.62% similarity) SFS68380.1 (75.24% identity, 86.67%similarity) APG04450.0 (75.16% identity, 87.58% similarity) APG08990.0(75.08% identity, 87.86% similarity) APG02403.0 26  27 28 Cry 97, 98,99, 100 98, 99, 100 APG00061.0 US_2016_0304898_A1-92 (96.22% identity,97.58% similarity) APG08931.0 (47.15% identity, 63.94% similarity)APG00206.0 US_2016_0304898_A1-199 (46.21% identity, 63.47% similarity)WP_048536348.1 (45.95% identity, 63.64% similarity) APG05034.0 (44.52%identity, 60.24% similarity) CA_2844913-198 (25.26% identity, 41.63%similarity) APG02438.0 29  30 Cry 90, 91, 92, 93, 94, 95, 92, 93, 94,95, 96, APG00094.0 US_2016_0177333_A1-50 (89.29% 96, 97, 98, 99, 100 97,98, 99, 100 identity, 91.96% similarity) APG07383.0 (85.57% identity,90.03% similarity) WP_064532221.1 (85.27% identity, 87.95% similarity)WP_063549999.1 (84.97% identity, 87.8% similarity) WP_074651312.1(68.18% identity, 78.25% similarity) APG00062.0 US_2016_0177333_A1-29(68.03% identity, 78.54% similarity) US_8759619_B2-24 (36.04% identity,52.88% similarity) US_8318900_B2-169 (35.84% identity, 52.27%similarity) US_8461421_B2-184 (34.63% identity, 52.16% similarity)APG00130.0 US_2016_0177333_A1-72 (33.61% identity, 51.26% similarity)APG00511.0 US_2016_0177333_A1-127 (33.47% identity, 51.26% similarity)APG00897.0 US_2016_0355842_A1-186 (33.01% identity, 48.74% similarity)WP_002187556.1 (32.9% identity, 46.84% similarity) APG01199.0 (32.61%identity, 49.93% similarity) APG06630.0 (32.47% identity, 48.22%similarity) US_8461421_B2-204 (32.17% identity, 46.43% similarity)APG00145.0 US_2016_0304898_A1-163 (31.62% identity, 46.44% similarity)US_8759619_B2-26 (31.38% identity, 49.8% similarity) APG00581.0US_2016_0355842_A1-133 (31.17% identity, 49.34% similarity) APG00041.0US_2016_0304898_A1-64 (30.64% identity, 46.08% similarity)WO_2015_118207-2 (30.12% identity, 43.93% similarity) US_8461421_B2-84(30.11% identity, 45.1% similarity) APG02572.0 31  32 Bin 75, 80, 85,90, 91, 92, 85, 90, 91, 92, 93, APG00988.0 (73.7% identity, 80.4%similarity) 93, 94, 95, 96, 97, 98, 94, 95, 96, 97, 98, APG00261.0US_2016_0311864_A1-100 99, 100 99, 100 (70.72% identity, 80.4%similarity) SDZ44649.1 (70.3% identity, 78.47% similarity) APG07247.0(69.23% identity, 80.15% similarity) WP_000727408.1 (69.14% identity,77.28% similarity) APG00648.0 AgB048P (68.24% identity, 78.66%similarity) CA_2844913-1 (68.15% identity, 75.8% similarity)CA_2844913-2 (68.15% identity, 75.8% similarity) WP_002187944.1 (67.99%identity, 80.4% similarity) WP_001258161.1 (67.74% identity, 80.4%similarity) CA_2844913-10 (67.74% identity, 79.9% similarity)WP_001258160.1 (67.49% identity, 80.15% similarity) APG00141.0US_2016_0311864_A1-34 (67.4% identity, 75.0% similarity) APG07491.0(67.31% identity, 75.3% similarity) EEL19841.1 (67.0% identity, 78.66%similarity) WP_002114997.1 (66.75% identity, 76.43% similarity)WP_070128649.1 (65.76% identity, 74.94% similarity) APG02630.0 33 34, 35Cry 90, 91, 92, 93, 94, 95, 90, 91, 92, 93, 94, APG04682.0 (85.94%identity, 89.99% similarity) 96, 97, 98, 99, 100 95, 96, 97, 98, 99,CA_2595901-4 (31.86% identity, 47.21% 100 similarity) US_8759619_B2-17(30.94% identity, 46.84% similarity) US_8759619_B2-3 (30.94% identity,46.84% similarity) CA_2595901-2 (30.93% identity, 46.14% similarity)APG00070.0 US_2016_0177333_A1-37 (30.3% identity, 45.06% similarity)US_8759619_B2-13 (30.3% identity, 44.73% similarity) US_8759619_B2-15(29.69% identity, 45.28% similarity) AEH76817.1 (29.12% identity, 45.31%similarity) US_8318900_B2-68 (29.03% identity, 45.49% similarity)AHI15916.1 (28.48% identity, 45.65% similarity) US_8318900_B2-18 (27.96%identity, 42.49% similarity) US_8318900_B2-67 (27.96% identity, 42.49%similarity) WP_061685895.1 (27.8% identity, 42.12% similarity)APG00651.0 (27.77% identity, 43.75% similarity) WP_050595403.1 (27.54%identity, 43.25% similarity) US_7923602_B2-6 (27.26% identity, 44.84%similarity) CA_2595901-15 (27.06% identity, 43.64% similarity)APG00524.0 (27.05% identity, 43.32% similarity) US_8461415_B2-25 (26.98%identity, 42.57% similarity) US_8461415_B2-58 (26.98% identity, 42.57%similarity) APG00083.0 US_2016_0304898_A1-111 (26.91% identity, 42.81%similarity) AHI15917.1 (26.57% identity, 42.1% similarity) APG00165.0US_2016_0355842_A1-11 (26.56% identity, 42.35% similarity) CA_2595901-11(26.55% identity, 43.03% similarity) US_5281530-4 (26.48% identity,42.97% similarity) US_5281530-3 (26.48% identity, 42.9% similarity)US_5670365_A-9 (26.44% identity, 42.34% similarity) US_5670365_A-9(26.44% identity, 42.34% similarity) US_5670365_A-8 (26.37% identity,42.34% similarity) US_5670365_A-8 (26.37% identity, 42.34% similarity)CA_2595901-13 (26.2% identity, 42.95% similarity) US_8461421_B2-81(26.18% identity, 42.65% similarity) US_2011_0214209_A1-6 (26.16%identity, 42.89% similarity) APG00703.0 (26.13% identity, 41.86%similarity) APG00052.0 US_2016_0177333_A1-25 (26.11% identity, 41.43%similarity) APG00052.0 US_2016_0177333_A1-25 (26.11% identity, 41.43%similarity) APG00217.0 US_2016_0355842_A1-24 (26.08% identity, 41.89%similarity) APG00622.0 (25.99% identity, 42.54% similarity) APG00622.0(25.99% identity, 42.54% similarity) US_7923602_B2-1 (25.97% identity,41.28% similarity) US_7923602_B2-1 (25.97% identity, 41.28% similarity)US_8318900_B2-17 (25.94% identity, 41.89% similarity) US_8318900_B2-66(25.94% identity, 41.89% similarity) APG00001.0 US_2016_0304898_A1-1(25.91% identity, 42.45% similarity) US_8147856_B2-32 (25.9% identity,41.17% similarity) US_8147856_B2-32 (25.9% identity, 41.17% similarity)US_7923602_B2-35 (25.76% identity, 43.61% similarity) US_7923602_B2-4(25.74% identity, 41.41% similarity) US_7923602_B2-13 (25.53% identity,41.51% similarity) US_5589382_A-4 (25.53% identity, 41.44% similarity)US_5959080_A-3 (25.47% identity, 41.26% similarity) APG00663.0 (25.44%identity, 42.13% similarity) US_8759619_B2-21 (25.22% identity, 40.57%similarity) APG00370.0 US_2016_0366881_A1-38 (25.05% identity, 40.08%similarity) APG02647.0 36  37 Cyt 85, 90, 91, 92, 93, 94, 90, 91, 92,93, 94, APG00462.0 US_2016_0311864_A1-183 95, 96, 97, 98, 99, 100 95,96, 97, 98, 99, (82.56% identity, 86.12% similarity) 100 APG08139.0(78.29% identity, 83.63% similarity) WP_043159001.1 (78.29% identity,83.63% similarity) WP_017413134.1 (76.95% identity, 81.91% similarity)WP_005311350.1 (76.95% identity, 81.56% similarity) WP_073536971.1(76.95% identity, 81.21% similarity) WP_058393986.1 (76.6% identity,81.56% similarity) WP_042469140.1 (76.41% identity, 81.34% similarity)WP_043151353.1 (76.41% identity, 80.99% similarity) WP_058407268.1(76.06% identity, 80.99% similarity) APG02701.0 38  39 MTX 85, 90, 91,92, 93, 94, 90, 91, 92, 93, 94, APG00585.0 US_2016_0366881_A1-116 95,96, 97, 98, 99, 100 95, 96, 97, 98, 99, (84.66% identity, 89.88%similarity) 100 WP_018673409.1 (83.13% identity, 89.57% similarity)APG06560.0 (81.6% identity, 88.34% similarity) APG00851.0US_2016_0311864_A1-222 (81.29% identity, 86.2% similarity)WP_061663532.1 (81.29% identity, 86.2% similarity) APG00427.0US_2016_0311864_A1-178 (80.67% identity, 88.34% similarity)WP_001039209.1 (69.7% identity, 80.3% similarity) APG01676.0 (66.17%identity, 76.65% similarity) A0B42285.1 (65.07% identity, 76.12%similarity) APG00309.0 US_2016_0355842_A1-56 (62.83% identity, 76.4%similarity) APG00989.0 (62.69% identity, 76.12% similarity) APG08411.0(60.9% identity, 75.22% similarity) APG02742.0 40  41 Bin 92, 93, 94,95, 96, 97, 97, 98, 99, 100 APG00731.0 US_2016_0311864_A1-211 98, 99,100 (91.69% identity, 96.25% similarity) APG00377.0US_2016_0311864_A1-154 (90.88% identity, 95.44% similarity) APG00669.0US_2016_0355842_A1-156 (89.81% identity, 95.44% similarity) APG00284.0US_2016_0311864_A1-117 (89.81% identity, 94.91% similarity) APG00035.0US_2016_0304898_A1-58 (89.01% identity, 95.71% similarity) APG00231.0US_2016_0311864_A1-86 (89.01% identity, 94.91% similarity)WP_000143307.1 (89.01% identity, 94.37% similarity) APG00356.0US_2016_0311864_A1-147 (88.74% identity, 94.37% similarity)WP_000143308.1 (87.94% identity, 94.1% similarity) APG00494.0 (87.67%identity, 94.64% similarity) APG00568.0 US_2016_0311864_A1-391 (87.67%identity, 94.64% similarity) APG00287.0 US_2016_0311864_A1-121 (87.67%identity, 93.3% similarity) APG00157.0 US_2016_0355842_A1-7 (87.13%identity, 93.3% similarity) WP_050845516.1 (86.6% identity, 92.76%similarity) APG00602.0 US_2016_0366881_A1-118 (86.6% identity, 91.69%similarity) AEX56523.1 (86.06% identity, 92.49% similarity) APG03055.042 43, 44 MTX 93, 94, 95, 96, 97, 98, 95, 96, 97, 98, 99, APG00940.0US_2016 0355842_A1-191 99, 100 100 (92.88% identity, 94.87% similarity)APG07058.0 (27.51% identity, 43.3% similarity) APG09864.0 (26.64%identity, 45.33% similarity) APG03101.0 45  46 MTX 70, 75, 80, 85, 90,91, 80, 85, 90, 91, 92, APG03324.0 (70.03% identity, 80.12% similarity)92, 93, 94, 95, 96, 97, 93, 94, 95, 96, 97, APG00323.0US_2016_0355842_A1-60 (69.44% 98, 99, 100 98, 99, 100 identity, 79.23%similarity) APG00586.0 US_2016_0355842_A1-135 (69.32% identity, 79.35%similarity) APG00420.0 US_2016_0355842_A1-85 (68.82% identity, 79.12%similarity) APG00860.0 US_2016_0355842_A1-180 (38.1% identity, 55.06%similarity) APG00718.0 (34.93% identity, 54.63% similarity)US_8318900_B2-77 (32.06% identity, 51.47% similarity) APG04586.0 (32.06%identity, 49.12% similarity) APG09100.0 (31.69% identity, 48.63%similarity) APG00645.0 US_2016_0355842_A1-151 (31.38% identity, 50.73%similarity) APG00425.0 US_2016_0355842_A1-87 (30.66% identity, 49.57%similarity) APG03147.0 47 Bin 45, 50, 55, 60, 65, 70, 60, 65, 70, 75,80, APG00880.0 US_2016 0366881_A1-134 75, 80, 85, 90, 91, 92, 85, 90,91, 92, 93, (42.36% identity, 55.23% similarity) 93, 94, 95, 96, 97, 98,94, 95, 96, 97, 98, WP_016093722.1 (41.71% identity, 56.42% 99, 100 99,100 similarity) APG00472.0 US_2016_0366881_A1-86 (41.58% identity,57.11% similarity) APG00642.0 US_2016_0311864_A1-202 (41.51% identity,57.44% similarity) APG00674.0 (41.51% identity, 56.92% similarity)WP_002167240.1 (41.42% identity, 55.94% similarity) APG00386.0US_2016_0311864_A1-158 (41.24% identity, 55.53% similarity) APG00837.0(40.75% identity, 55.5% similarity) APG05284.0 (40.74% identity, 58.47%similarity) APG00748.0 US_2016_0311864_A1-214 (39.41% identity, 58.71%similarity) APG00215.0 US_2016_0311864_A1-78 (38.87% identity, 54.69%similarity) WP_048517129.1 (38.03% identity, 55.05% similarity)WP_064016909.1 (37.77% identity, 51.6% similarity) WP_002090518.1 (37.5%identity, 55.32% similarity) WP_042162506.1 (37.43% identity, 55.76%similarity) APG06051.0 (37.17% identity, 49.47% similarity)WP_002016877.1 (37.07% identity, 54.93% similarity) WP_052728656.1(36.39% identity, 53.4% similarity) WP_016097060.1 (36.2% identity,52.6% similarity) US_9238823_B2-7 (35.36% identity, 52.51% similarity)APG03187.0 48  49 Bin 92, 93, 94, 95, 96, 97, 97, 98, 99, 100 APG00403.0US_2016_0355842_A1-78 (91.19% 98, 99, 100 identity, 96.11% similarity)US_8461421_B2-5 (51.01% identity, 65.08% similarity) US_8461421_B2-65(51.01% identity, 65.08% similarity) US_8461421_B2-146 (43.56% identity,55.15% similarity) WP_065486893.1 (31.11% identity, 51.11% similarity)US_8461421_B2-67 (28.5% identity, 46.79% similarity) APG05259.0 (28.5%identity, 44.89% similarity) APG00256.0 US_2016_0355842_A1-41 (28.22%identity, 48.02% similarity) US_8461421_B2-4 (27.73% identity, 43.86%similarity) US_8461421_B2-64 (27.73% identity, 43.86% similarity)WP_048517129.1 (27.54% identity, 43.48% similarity) APG03147.0 (27.45%identity, 43.2% similarity) APG00880.0 US_2016_0366881_A1-134 (27.45%identity, 42.16% similarity) WP_002090518.1 (27.29% identity, 43.24%similarity) US_8461421_B2-6 (27.11% identity, 42.37% similarity)US_8461421_B2-66 (27.11% identity, 42.37% similarity) APG00386.0US_2016_0311864_A1-158 (26.85% identity, 45.57% similarity) APG00215.0US_2016_0311864_A1-78 (26.65% identity, 44.01% similarity)US_8461421_B2-148 (26.62% identity, 40.53% similarity) APG00837.0(26.03% identity, 41.12% similarity) WP_016097060.1 (26.01% identity,39.46% similarity) WP_016093722.1 (26.0% identity, 40.28% similarity)WP_042162506.1 (25.59% identity, 42.18% similarity) US_9238823_B2-7(25.37% identity, 42.61% similarity) WP_065486894.1 (25.28% identity,40.35% similarity) APG03195.0 50 51, 52 MTX 85, 90, 91, 92, 93, 94, 90,91, 92, 93, 94, SFS68380.1 (86.94% identity, 90.5% similarity) 95, 96,97, 98, 99, 100 95, 96, 97, 98, 99, APG07049.0 (82.49% identity, 87.24%similarity) 100 APG05969.0 (82.2% identity, 87.54% similarity)APG05084.0 (82.2% identity, 86.65% similarity) WP_025150761.1 (82.2%identity, 86.65% similarity) APG04485.0 (81.6% identity, 88.13%similarity) APG02921.0 (81.6% identity, 85.46% similarity) APG04226.0(81.01% identity, 87.24% similarity) APG06989.0 (81.01% identity, 85.76%similarity) APG01536.0 (80.71% identity, 86.05% similarity) APG08990.0(80.71% identity, 85.76% similarity) APG03279.0 53 Bin 75, 80, 85, 90,91, 92, 85, 90, 91, 92, 93, WP_017154552.1 (78.72% identity, 86.44% 93,94, 95, 96, 97, 98, 94, 95, 96, 97, 98, similarity) 99, 100 99, 100US_9321814_B2-3 (74.73% identity, 82.71% similarity) US_9321814_B2-2(74.54% identity, 82.49% similarity) APG00596.0 US_2016_0311864_A1-196(74.21% identity, 82.63% similarity) US_9321814_B2-4 (73.4% identity,81.38% similarity) US_9321814_B2-5 (72.34% identity, 80.32% similarity)APG00741.0 US_2016_0311864_A1-213 (71.09% identity, 79.58% similarity)APG00413.0 US_2016_0311864_A1-174 (70.78% identity, 81.5% similarity)APG00230.0 US_2016_0311864_A1-85 (69.84% identity, 78.84% similarity)WP_050001305.1 (68.88% identity, 79.26% similarity) APG00757.0US_2016_0311864_A1-215 (66.92% identity, 75.87% similarity) APG00482.0US_2016_0366881_A1-94 (65.92% identity, 74.63% similarity) APG03324.0 54 55 MTX 90, 91, 92, 93, 94, 95, 92, 93, 94, 95, 96, APG00420.0US_2016_0355842_A1-85 (88.24% 96, 97, 98, 99, 100 97, 98, 99, 100identity, 91.76% similarity) APG00586.0 US_2016_0355842_A1-135 (87.32%identity, 90.27% similarity) APG00323.0 US_2016_0355842_A1-60 (86.94%identity, 90.21% similarity) APG03101.0 (70.03% identity, 80.12%similarity) APG00860.0 US_2016_0355842_A1-180 (42.52% identity, 58.94%similarity) APG00718.0 (40.06% identity, 58.75% similarity) APG04586.0(36.78% identity, 54.6% similarity) US_8318900_B2-77 (35.67% identity,55.34% similarity) APG09100.0 (33.61% identity, 53.28% similarity)APG00425.0 US_2016_0355842_A1-87 (32.35% identity, 52.65% similarity)APG00645.0 US_2016_0355842_A1-151 (31.87% identity, 54.68% similarity)APG03498.0 56  57 Cry 75, 80, 85, 90, 91, 92, 85, 90, 91, 92, 93,APG00504.0 US_2016_0366881_A1-99 (71.87% 93, 94, 95, 96, 97, 98, 94, 95,96, 97, 98, identity, 82.09% similarity) 99, 100 99, 100 APG07889.0(57.67% identity, 68.17% similarity) APG07679.0 (56.04% identity, 65.71%similarity) US_8759619_B2-25 (55.98% identity, 65.02% similarity)US_8759619_B2-25 (55.98% identity, 65.02% similarity) APG00430.0US_2016_0366881_A1-54 (54.34% identity, 66.54% similarity)US_8461421_B2-70 (54.18% identity, 65.58% similarity) APG00974.0 (53.8%identity, 64.06% similarity) US_2016_0122399_A1-2 (53.35% identity,64.48% similarity) US_2016_0122399_A1-4 (53.32% identity, 64.45%similarity) APG00068.0 US_2016_0177333_A1-35 (53.31% identity, 63.46%similarity) US_2016_0122399_A1-1 (53.28% identity, 64.41% similarity)APG00056.0 US_2016_0311864_A1-1 (52.83% identity, 64.91% similarity)ADO32760.1 (52.78% identity, 64.2% similarity) US_8461421_B2-98 (52.75%identity, 63.07% similarity) US_8461421_B2-91 (52.59% identity, 63.31%similarity) US_8461421_B2-91 (52.59% identity, 63.31% similarity)APG01387.0 (52.46% identity, 64.29% similarity) ADO32759.1 (52.32%identity, 64.2% similarity) APG00469.0 US_2016_0366881_A1-82 (52.03%identity, 63.68% similarity) APG06739.0 (52.0% identity, 62.55%similarity) APG06739.0 (52.0% identity, 62.55% similarity)CA_2866166-324 (51.99% identity, 63.88% similarity) APG05370.0 (51.85%identity, 62.61% similarity) APG03519.0 (51.56% identity, 62.83%similarity) APG00310.0 US_2016_0366881_A1-4 (51.52% identity, 62.67%similarity) US_8318900_B2-38 (51.5% identity, 63.04% similarity)US_8318900_B2-87 (51.5% identity, 63.04% similarity) APG00710.0 (51.35%identity, 63.05% similarity) APG00058.0 US_2016_0311864_A1-3 (51.03%identity, 62.64% similarity) APG01329.0 (50.87% identity, 62.57%similarity) US_8759619_B2-23 (50.72% identity, 62.45% similarity)US_8759619_B2-6 (50.72% identity, 62.45% similarity) APG05930.0 (50.6%identity, 60.76% similarity) US_8759619_B2-19 (50.59% identity, 62.72%similarity) US_8461421_B2-73 (50.42% identity, 63.19% similarity)US_8318900_B2-82 (50.18% identity, 61.79% similarity) US_8318900_B2-82(50.18% identity, 61.79% similarity) US_8318900_B2-88 (50.15% identity,60.38% similarity) US_8318900_B2-88 (50.15% identity, 60.38% similarity)APG07870.0 (50.07% identity, 61.44% similarity) APG09604.0 (50.04%identity, 60.1% similarity) APG03889.0 58  59 MTX 93, 94, 95, 96, 97,98, 95, 96, 97, 98, 99, APG00429.0 US_2016_0366881_A1-53 (92.39% 99, 100100 identity, 94.65% similarity) APG00994.0 US_2016_0355842_A1-198(59.23% identity, 73.83% similarity) APG02557.0 (57.14% identity, 72.53%similarity) APG03900.0 60 MTX 90, 91, 92, 93, 94, 95, 93, 94, 95, 96,97, APG00218.0 US_2016_0355842_A1-26 (88.89% 96, 97, 98, 99, 100 98, 99,100 identity, 92.4% similarity) APG00245.0 US_2016_0355842_A1-34 (88.01%identity, 91.52% similarity) APG01189.0 (56.23% identity, 69.57%similarity) APG00187.0 US_2016_0355842_A1-20 (55.65% identity, 71.01%similarity) US_8829279_B2-42 (31.43% identity, 50.0% similarity)APG04069.0 61  62 Vip4 70, 75, 80, 85, 90, 91, 80, 85, 90, 91, 92,CA_2844913-129 (66.87% identity, 78.42% 92, 93, 94, 95, 96, 97, 93, 94,95, 96, 97, similarity) 98, 99, 100 98, 99, 100 CA_2844913-130 (66.87%identity, 78.42% similarity) WP_016123960.1 (65.5% identity, 75.97%similarity) AGT29561.1 (54.52% identity, 70.23% similarity) APG01474.0(54.15% identity, 69.74% similarity) SCC34829.1 (53.11% identity, 66.77%similarity) APG00106.0 US_2016_0304898_A1-134 (51.64% identity, 64.83%similarity) US_2016_0339078_A1-29674 (51.27% identity, 69.18%similarity) APG08992.0 (51.09% identity, 65.84% similarity) APG07526.0(50.97% identity, 67.24% similarity) APG01927.0 (50.75% identity, 64.49%similarity) APG09984.0 (50.71% identity, 65.35% similarity) APG04931.0(50.05% identity, 66.84% similarity) APG04345.0 63  64 MTX 96, 97, 98,99, 100 97, 98, 99, 100 APG00686.0 US_2016_0355842_A1-159 (95.2%identity, 96.4% similarity) APG00860.0 US_2016_0355842_A1-180 (28.77%identity, 45.87% similarity) APG00718.0 (28.05% identity, 46.74%similarity) APG03324.0 (26.82% identity, 46.65% similarity) APG00586.0US_2016_0355842_A1-135 (26.17% identity, 44.35% similarity) APG00420.0US_2016_0355842_A1-85 (25.82% identity, 43.13% similarity) APG00323.0US_2016_0355842_A1-60 (25.62% identity, 43.8% similarity) APG03101.0(25.49% identity, 42.58% similarity) APG04586.0 (25.42% identity, 44.69%similarity) APG04415.0 65 Cry 85, 90, 91, 92, 93, 94, 90, 91, 92, 93,94, APG00087.0 US_2016_0177333_A1-47 (82.83% 95, 96, 97, 98, 99, 100 95,96, 97, 98, 99, identity, 88.55% similarity) 100 APG00189.0US_2016_0311864_A1-59 (82.57% identity, 88.27% similarity) APG08396.0(82.09% identity, 87.64% similarity) APG00179.0 US_2016_0304898_A1-186(36.06% identity, 53.02% similarity) US_2016_0017363_A1-27 (36.06%identity, 53.02% similarity) US_2016_0017363_A1-28 (33.88% identity,48.74% similarity) US_2016_0017363_A1-29 (33.73% identity, 48.59%similarity) US_2016_0017363_A1-30 (33.58% identity, 48.44% similarity)US_2016_0017363_A1-31 (33.28% identity, 47.85% similarity)US_8318900_B2-169 (31.44% identity, 47.03% similarity)KR_1020000046327-1 (31.34% identity, 46.87% similarity)KR_1020130140355-3 (31.21% identity, 46.67% similarity) CA_2309131-16(31.19% identity, 46.78% similarity) US_6025545_A-5 (31.19% identity,46.78% similarity) CA_2234656-1 (31.1% identity, 46.65% similarity)US_8759619_B2-24 (30.5% identity, 45.68% similarity) APG04586.0 66 MTX90, 91, 92, 93, 94, 95, 93, 94, 95, 96, 97, US_8318900_B2-77 (89.6%identity, 92.35% 96, 97, 98, 99, 100 98, 99, 100 similarity) APG09100.0(49.01% identity, 63.66% similarity) APG00860.0 US_2016_0355842_A1-180(42.04% identity, 55.26% similarity) APG00425.0 US_2016_0355842_A1-87(40.0% identity, 57.88% similarity) APG00718.0 (40.0% identity, 55.76%similarity) APG00645.0 US_2016_0355842_A1-151 (39.09% identity, 59.39%similarity) APG00586.0 US_2016_0355842_A1-135 (36.86% identity, 54.0%similarity) APG03324.0 (36.78% identity, 54.6% similarity) APG00420.0US_2016_0355842_A1-85 (36.75% identity, 53.85% similarity) APG00323.0US_2016_0355842_A1-60 (36.21% identity, 54.89% similarity) APG04671.0 67 68 69 Cry 65, 70, 75, 80, 85, 90, 75, 80, 85, 90, 91, AEX56526.1(68.18% identity, 78.1% similarity) 91, 92, 93, 94, 95, 96, 92, 93, 94,95, 96, CA_2516349-26 (64.43% identity, 73.14% 97, 98, 99, 100 97, 98,99, 100 similarity) CA_2516349-27 (64.29% identity, 73.14% similarity)CA_2516349-29 (64.0% identity, 72.71% similarity) APG00204.0US_2016_0177333_A1-89 (61.39% identity, 72.42% similarity) CA_2516349-25(60.78% identity, 69.0% similarity) APG03786.0 (58.45% identity, 69.91%similarity) CA_2866166-428 (53.79% identity, 66.09% similarity)APG00695.0 (52.86% identity, 67.2% similarity) AIN76756.1 (51.9%identity, 66.18% similarity) CA_2866166-374 (50.8% identity, 65.7%similarity) CA_2516349-51 (50.51% identity, 64.36% similarity)APG04699.0 70 MTX 85, 90, 91, 92, 93, 94, 90, 91, 92, 93, 94, APG05328.0(80.59% identity, 81.18% similarity) 95, 96, 97, 98, 99, 100 95, 96, 97,98, 99, APG00559.0 US_2016_0311864_A1-191 100 (78.38% identity, 85.14%similarity) APG00067.0 US_2016_0311864_A1-7 (77.85% identity, 86.24%similarity) APG00408.0 US_2016_0311864_A1-171 (75.84% identity, 84.9%similarity) APG00954.0 (75.83% identity, 84.44% similarity) APG08589.0(69.59% identity, 83.45% similarity) APG07655.0 (69.59% identity, 79.73%similarity) APG01506.0 (69.26% identity, 80.41% similarity) APG00155.0US_2016_0311864_A1-40 (69.26% identity, 79.05% similarity) APG09682.0(69.02% identity, 81.48% similarity) APG08088.0 (68.56% identity, 80.6%similarity) APG00260.0 US_2016_0355842_A1-44 (67.76% identity, 78.29%similarity) CA_2844913-100 (67.57% identity, 80.41% similarity)APG07639.0 (67.57% identity, 80.07% similarity) APG06690.0 (67.23%identity, 81.08% similarity) APG08241.0 (67.22% identity, 78.81%similarity) APG04720.0 (67.0% identity, 78.55% similarity) APG01245.0US_2016_0311864_A1-398 (66.89% identity, 80.74% similarity) APG00006.0US_2016_0304898_A1-9 (66.67% identity, 77.99% similarity) APG00036.0US_2016_0304898_A1-60 (66.67% identity, 77.23% similarity) APG00345.0US_2016_0366881_A1-23 (66.55% identity, 81.08% similarity) APG00201.0US_2016_0304898_A1-193 (66.45% identity, 77.63% similarity) APG00930.0US_2016_0311864_A1-397 (66.44% identity, 80.87% similarity) APG08718.0(66.23% identity, 78.15% similarity) WP_000963933.1 (66.23% identity,78.15% similarity) APG00107.0 US_2016_0311864_A1-21 (66.22% identity,79.39% similarity) APG00749.0 (66.12% identity, 77.63% similarity)APG02638.0 (66.01% identity, 79.21% similarity) APG00589.0 (65.89%identity, 79.8% similarity) APG03379.0 (65.89% identity, 78.81%similarity) APG00847.0 US_2016_0304898_A1-223 (65.79% identity, 77.63%similarity) APG00137.0 US_2016_0311864_A1-33 (65.58% identity, 78.25%similarity) APG00898.0 US_2016_0311864_A1-224 (65.02% identity, 78.88%similarity) APG04912.0 71 PI- 60, 65, 70, 75, 80, 85, 70, 75, 80, 85,90, WP_063549551.1 (58.32% identity, 71.04% PLC 90, 91, 92, 93, 94, 95,91, 92, 93, 94, 95, similarity) 96, 97, 98, 99, 100 96, 97, 98, 99, 100APG00078.0 US_2016_0366881_A1-1 (57.33% identity, 69.35% similarity)WP_036684392.1 (49.93% identity, 62.38% similarity) WP_016123021.1(48.63% identity, 62.35% similarity) APG00338.0 US_2016_0366881_A1-21(44.18% identity, 55.56% similarity) WP_016084067.1 (40.35% identity,56.46% similarity) WP_000836979.1 (39.0% identity, 54.94% similarity)EEM93103.1 (38.52% identity, 54.85% similarity) APG00572.0 (31.4%identity, 43.8% similarity) APG00738.0 (28.78% identity, 38.15%similarity) APG00732.0 US_2016_0311864_A1-212 (28.39% identity, 38.97%similarity) WP_060751696.1 (28.15% identity, 39.01% similarity)WP_060751696.1 (28.15% identity, 39.01% similarity) APG00638.0 (27.77%identity, 38.18% similarity) APG06043.0 (27.74% identity, 38.84%similarity) APG06043.0 (27.74% identity, 38.84% similarity) EOQ00432.1(27.5% identity, 38.78% similarity) WP_001033587.1 (27.07% identity,37.84% similarity) WP_001033587.1 (27.07% identity, 37.84% similarity)US_8829279_B2-9 (26.73% identity, 37.58% similarity) US_8318900_B2-79(26.32% identity, 37.5% similarity) APG05259.0 72  73 Bin 70, 75, 80,85, 90, 91, 85, 90, 91, 92, 93, US_8461421_B2-6 (68.58% identity, 81.3%92, 93, 94, 95, 96, 97, 94, 95, 96, 97, 98, similarity) 98, 99, 100 99,100 US_8461421_B2-66 (68.58% identity, 81.3% similarity)US_8461421_B2-148 (65.97% identity, 77.75% similarity) APG06801.0(54.91% identity, 57.68% similarity) WP_065486894.1 (42.36% identity,61.9% similarity) US_8461421_B2-4 (33.33% identity, 51.64% similarity)US_8461421_B2-64 (33.33% identity, 51.64% similarity) WP_065486893.1(31.49% identity, 49.62% similarity) APG00256.0 US_2016_0355842_A1-41(30.35% identity, 49.25% similarity) US_8461421_B2-67 (29.77% identity,51.4% similarity) APG03187.0 (28.5% identity, 44.89% similarity)US_8461421_B2-5 (27.11% identity, 44.78% similarity) US_8461421_B2-65(27.11% identity, 44.78% similarity) US_8461421_B2-144 (26.98% identity,40.1% similarity) APG00403.0 US_2016_0355842_A1-78 (26.85% identity,43.52% similarity) APG00880.0 US_2016_0366881_A1-134 (25.73% identity,41.26% similarity) APG03147.0 (25.71% identity, 39.76% similarity)WP_052728656.1 (25.48% identity, 41.43% similarity) US_9238823_B2-7(25.12% identity, 42.79% similarity) APG05284.0 74 Bin 80, 85, 90, 91,92, 93, 90, 91, 92, 93, 94, APG00472.0 US_2016_0366881_A1-86 (77.08% 94,95, 96, 97, 98, 99, 95, 96, 97, 98, 99, identity, 85.96% similarity) 100100 APG00674.0 (72.49% identity, 83.95% similarity) APG00642.0US_2016_0311864_A1-202 (70.0% identity, 82.0% similarity) APG03147.0(40.74% identity, 58.47% similarity) APG00880.0 US_2016_0366881_A1-134(36.41% identity, 51.63% similarity) APG00215.0 US_2016_0311864_A1-78(34.73% identity, 51.54% similarity) WP_029439068.1 (34.66% identity,48.94% similarity) APG00837.0 (34.46% identity, 50.65% similarity)WP_016093722.1 (33.79% identity, 49.59% similarity) APG00591.0US_2016_0311864_A1-194 (33.24% identity, 50.4% similarity) APG00440.0US_2016_0366881_A1-65 (32.89% identity, 48.42% similarity) APG00386.0US_2016_0311864_A1-158 (32.8% identity, 52.15% similarity) APG00415.0US_2016_0311864_A1-175 (32.78% identity, 51.24% similarity)WP_028595059.1 (32.57% identity, 49.11% similarity) APG00550.0US_2016_0311864_A1-190 (32.32% identity, 52.49% similarity) APG00314.0US_2016_0311864_A1-136 (32.17% identity, 45.64% similarity)WP_033679178.1 (31.9% identity, 50.89% similarity) WP_029439066.1(31.83% identity, 50.99% similarity) APG00806.0 (30.81% identity, 48.66%similarity) APG04176.0 (30.36% identity, 48.19% similarity)WP_000839920.1 (30.35% identity, 48.76% similarity) APG05715.0 (30.22%identity, 47.96% similarity) CA_2844913-146 (30.15% identity, 48.77%similarity) APG00011.0 US_2016_0304898_A1-17 (30.15% identity, 46.91%similarity) APG00716.0 US_2016_0311864_A1-393 (30.12% identity, 47.9%similarity) APG00376.0 US_2016_0311864_A1-153 (30.02% identity, 48.88%similarity) APG09768.0 (30.02% identity, 48.88% similarity) APG05311.075  76 Cry 90, 91, 92, 93, 94, 95, 90, 91, 92, 93, 94, APG03519.0(86.25% identity, 89.65% similarity) 96, 97, 98, 99, 100 95, 96, 97, 98,99, JP_2002_335967-2 (73.86% identity, 80.72% 100 similarity)CA_2866166-327 (73.58% identity, 80.26% similarity) US_8461421_B2-70(71.86% identity, 78.47% similarity) APG07889.0 (69.01% identity, 76.83%similarity) APG09604.0 (55.85% identity, 63.22% similarity) APG09604.0(55.85% identity, 63.22% similarity) APG00710.0 (55.26% identity, 66.27%similarity) US_8318900_B2-38 (54.51% identity, 65.8% similarity)US_8318900_B2-87 (54.32% identity, 65.19% similarity) US_8759619_B2-6(53.89% identity, 65.38% similarity) APG00056.0 US_2016_0311864_A1-1(53.8% identity, 63.85% similarity) US_8759619_B2-23 (53.7% identity,64.78% similarity) APG01329.0 (53.31% identity, 65.54% similarity)APG00430.0 US_2016_0366881_A1-54 (52.87% identity, 63.31% similarity)APG05370.0 (52.7% identity, 63.58% similarity) ADO32760.1 (52.46%identity, 62.44% similarity) US_8318900_B2-82 (52.4% identity, 62.7%similarity) US_8318900_B2-82 (52.4% identity, 62.7% similarity)US_8461421_B2-72 (52.15% identity, 63.99% similarity) APG00504.0US_2016_0366881_A1-99 (51.92% identity, 61.2% similarity) APG01387.0(51.7% identity, 63.28% similarity) ADO32759.1 (51.6% identity, 62.87%similarity) CA_2866166-324 (51.6% identity, 62.87% similarity)APG06650.0 (51.3% identity, 63.19% similarity) APG00974.0 (51.29%identity, 60.89% similarity) APG00974.0 (51.29% identity, 60.89%similarity) APG07870.0 (51.28% identity, 62.93% similarity) APG00068.0US_2016_0177333_A1-35 (50.95% identity, 62.19% similarity) APG00054.0US_2016_0304898_A1-84 (50.91% identity, 60.87% similarity)US_2016_0017363_A1-5 (50.91% identity, 60.87% similarity) APG00469.0US_2016_0366881_A1-82 (50.8% identity, 62.21% similarity)US_8318900_B2-81 (50.63% identity, 61.96% similarity) APG00058.0US_2016_0311864_A1-3 (50.61% identity, 62.03% similarity) APG00687.0(50.49% identity, 62.38% similarity) APG06739.0 (50.45% identity, 60.25%similarity) APG06739.0 (50.45% identity, 60.25% similarity)US_8759619_B2-27 (50.17% identity, 61.31% similarity) APG05341.0 77  78MTX 75, 80, 85, 90, 91, 92, 85, 90, 91, 92, 93, APG00324.0US_2016_0355842_A1-61 (71.21% 93, 94, 95, 96, 97, 98, 94, 95, 96, 97,98, identity, 81.75% similarity) 99, 100 99, 100 APG00253.0US_2016_0355842_A1-39 (48.05% identity, 61.3% similarity) APG00764.0US_2016_0355842_A1-168 (47.19% identity, 62.5% similarity) APG00322.0US_2016_0355842_A1-59 (46.82% identity, 61.32% similarity) APG00020.0US_2016_0304898_A1-33 (46.75% identity, 60.52% similarity) APG00481.0US_2016_0355842_A1-103 (46.68% identity, 60.71% similarity) APG01508.0(46.49% identity, 60.52% similarity) APG08278.0 (46.23% identity, 61.06%similarity) APG00959.0 US_2016_0355842_A1-194 (46.06% identity, 61.07%similarity) APG00418.0 US_2016_0355842_A1-84 (45.71% identity, 59.74%similarity) APG00646.0 US_2016_0355842_A1-201 (45.57% identity, 60.0%similarity) APG00635.0 US_2016_0311864_A1-201 (45.55% identity, 60.56%similarity) APG00268.0 US_2016_0355842_A1-45 (45.48% identity, 60.55%similarity) APG00393.0 US_2016_0355842_A1-76 (45.2% identity, 61.11%similarity) APG05669.0 79 MAC 30, 35, 40, 45, 50, 55, 50, 55, 60, 65,70, WP_046018755.1 (31.46% identity, 49.7% PF 60, 65, 70, 75, 80, 85,75, 80, 85, 90, 91, similarity) 90, 91, 92, 93, 94, 95, 92, 93, 94, 95,96, APG02912.0 (29.22% identity, 46.3% similarity) 96, 97, 98, 99, 10097, 98, 99, 100 WP_019961352.1 (28.99% identity, 50.3% similarity)APG03726.0 (28.35% identity, 47.38% similarity) SHO57735.1 (28.3%identity, 47.99% similarity) OIP63138.1 (27.32% identity, 42.13%similarity) WP_073439185.1 (27.18% identity, 47.62% similarity)US_9394345_B2-820 (26.82% identity, 44.25% similarity) APG07857.0(25.96% identity, 45.0% similarity) WP_049911819.1 (25.38% identity,43.23% similarity) US_8575425_B2-15 (25.18% identity, 37.95% similarity)APG05886.0 80  81 Cry 85, 90, 91, 92, 93, 94, 90, 91, 92, 93, 94,APG00034.0 US_2016_0177333_A1-14 (84.18% 95, 96, 97, 98, 99, 100 95, 96,97, 98, 99, identity, 88.19% similarity) 100 APG00383.0 (64.62%identity, 76.1% similarity) APG00101.0 US_2016_0177333_A1-57 (64.0%identity, 75.03% similarity) APG00608.0 (61.98% identity, 71.54%similarity) APG00002.0 US_2016_0177333_A1-1 (59.39% identity, 69.27%similarity) APG00048.0 US_2016_0177333_A1-22 (52.02% identity, 65.17%similarity) WP_065482678.1 (50.17% identity, 63.14% similarity)US_5596071_A-10 (38.24% identity, 54.11% similarity) WP_065485204.1(37.69% identity, 54.45% similarity) US_7923602_B2-27 (37.37% identity,53.03% similarity) US_7923602_B2-20 (37.34% identity, 52.81% similarity)US_7923602_B2-21 (37.34% identity, 52.81% similarity)US_2011_0231963_A1-9 (36.74% identity, 51.17% similarity)US_7923602_B2-7 (36.63% identity, 51.16% similarity) US_7923602_B2-8(36.63% identity, 51.16% similarity) CA_2840683-3 (36.59% identity,51.92% similarity) APG00097.0 US_2016_0177333_A1-53 (36.34% identity,51.68% similarity) US_7923602_B2-24 (36.31% identity, 51.55% similarity)US_7923602_B2-25 (36.31% identity, 51.55% similarity) US_7923602_B2-31(36.29% identity, 51.39% similarity) US_9139843_B2-3 (36.29% identity,51.34% similarity) APG00497.0 US_2016_0177333_A1-125 (36.26% identity,50.99% similarity) US_7923602_B2-29 (36.17% identity, 51.22% similarity)US_7923602_B2-19 (36.08% identity, 50.52% similarity) US_7923602_B2-22(35.66% identity, 50.0% similarity) US_7923602_B2-10 (35.59% identity,49.77% similarity) US_7923602_B2-23 (35.58% identity, 49.88% similarity)US_7923602_B2-12 (35.47% identity, 49.61% similarity) US_9139843_B2-5(35.45% identity, 50.23% similarity) US_7923602_B2-15 (35.34% identity,49.5% similarity) APG05896.0 82 MTX 50, 55, 60, 65, 70, 75, 65, 70, 75,80, 85, APG06242.0 (50.92% identity, 64.64% similarity) 80, 85, 90, 91,92, 93, 90, 91, 92, 93, 94, APG09338.0 (49.87% identity, 60.45%similarity) 94, 95, 96, 97, 98, 99, 95, 96, 97, 98, 99, US_8829279_B2-11(48.19% identity, 61.84% 100 100 similarity) APG00051.0US_2016_0304898_A1-81 (47.37% identity, 60.4% similarity) APG05372.0(47.13% identity, 62.42% similarity) APG00911.0 US_2016_0355842_A1-189(42.63% identity, 56.58% similarity) APG00854.0 US_2016_0355842_A1-179(42.11% identity, 56.58% similarity) APG09211.0 (41.67% identity, 53.68%similarity) APG00293.0 US_2016_0355842_A1-51 (38.35% identity, 49.37%similarity) APG00170.0 US_2016_0355842_A1-13 (36.78% identity, 47.84%similarity) APG06043.0 83  84 PI- 40, 45, 50, 55, 60, 65, 55, 60, 65,70, 75, WP_002151139.1 (68.42% identity, 81.05% PLC 70, 75, 80, 85, 90,91, 80, 85, 90, 91, 92, similarity) 92, 93, 94, 95, 96, 97, 93, 94, 95,96, 97, WP_002016970.1 (67.79% identity, 80.84% 98, 99, 100 98, 99, 100similarity) WP_002169683.1 (67.79% identity, 80.63% similarity)WP_071745347.1 (67.79% identity, 80.63% similarity) WP_016097117.1(67.58% identity, 80.84% similarity) WP_063218360.1 (67.58% identity,80.63% similarity) WP_016092549.1 (67.58% identity, 80.42% similarity)WP_071770677.1 (67.37% identity, 80.63% similarity) WP_002069636.1(67.37% identity, 80.42% similarity) WP_002090640.1 (67.16% identity,80.21% similarity) APG06187.0 85  86 MTX 55, 60, 65, 70, 75, 80, 75, 80,85, 90, 91, WP_019421057.1 (54.27% identity, 71.65% 85, 90, 91, 92, 93,94, 92, 93, 94, 95, 96, similarity) 95, 96, 97, 98, 99, 100 97, 98, 99,100 APG02408.0 (52.84% identity, 70.15% similarity) WP_016084062.1(51.52% identity, 71.52% similarity) APG02293.0 (51.52% identity, 70.61%similarity) APG04640.0 (51.35% identity, 69.97% similarity)WP_016085044.1 (51.21% identity, 71.21% similarity) WP_050845726.1(51.21% identity, 70.3% similarity) WP_016084449.1 (50.91% identity,70.91% similarity) WP_000823322.1 (50.61% identity, 70.0% similarity)APG04804.0 (50.3% identity, 70.78% similarity) WP_016083901.1 (50.3%identity, 69.39% similarity) WP_016085279.1 (50.3% identity, 69.39%similarity) WP_016084436.1 (50.0% identity, 69.09% similarity)APG06228.0 87  88 MTX 90, 91, 92, 93, 94, 95, 91, 92, 93, 94, 95,APG00980.0 (88.92% identity, 90.12% similarity) 96, 97, 98, 99, 100 96,97, 98, 99, 100 US_8318900_B2-78 (64.11% identity, 75.77% similarity)APG00590.0 US_2016_0311864_A1-193 (60.3% identity, 72.84% similarity)ANN35739.1 (58.59% identity, 73.31% similarity) CA_2844913-102 (58.46%identity, 73.23% similarity) APG09155.0 (57.98% identity, 73.01%similarity) APG09055.0 (57.31% identity, 71.64% similarity) APG00146.0US_2016_0304898_A1-166 (57.14% identity, 72.98% similarity) APG00387.0US_2016_0366881_A1-45 (56.8% identity, 71.0% similarity) WP_000794514.1(56.52% identity, 73.6% similarity) APG00351.0 US_2016_0366881_A1-28(56.52% identity, 69.86% similarity) APG09492.0 (56.23% identity, 69.57%similarity) APG06242.0 89 90, 91 MTX 65, 70, 75, 80, 85, 90, 75, 80, 85,90, 91, APG09338.0 (62.9% identity, 74.45% similarity) 91, 92, 93, 94,95, 96, 92, 93, 94, 95, 96, US_8829279_B2-11 (61.74% identity, 72.82%97, 98, 99, 100 97, 98, 99, 100 similarity) APG00051.0US_2016_0304898_A1-81 (52.87% identity, 70.57% similarity) APG09211.0(51.67% identity, 64.52% similarity) APG05896.0 (50.92% identity, 64.64%similarity) APG00911.0 US_2016_0355842_A1-189 (50.65% identity, 63.57%similarity) APG05372.0 (48.93% identity, 59.36% similarity) APG00854.0US_2016_0355842_A1-179 (48.61% identity, 61.01% similarity) APG00293.0US_2016_0355842_A1-51 (47.1% identity, 58.45% similarity) APG00170.0US_2016_0355842_A1-13 (46.43% identity, 58.33% similarity) APG06401.0 92 93 Bin 55, 60, 65, 70, 75, 80, 70, 75, 80, 85, 90, APG00075.0US_2016_0177333_A1-40 (54.67% 85, 90, 91, 92, 93, 94, 91, 92, 93, 94,95, identity, 67.12% similarity) 95, 96, 97, 98, 99, 100 96, 97, 98, 99,100 APG00750.0 US_2016_0366881_A1-132 (40.67% identity, 52.78%similarity) US_7692068_B2-2 (39.88% identity, 55.49% similarity)APG00734.0 US_2016_0366881_A1-131 (39.59% identity, 51.49% similarity)APG00706.0 (38.18% identity, 51.74% similarity) US_6555655_B1-14 (38.18%identity, 50.18% similarity) CA_2866166-398 (37.62% identity, 55.45%similarity) CA_2866166-394 (36.59% identity, 53.82% similarity)APG00063.0 US_2016_0304898_A1-95 (35.9% identity, 46.99% similarity)APG00629.0 US_2016_0366881_A1-126 (32.93% identity, 44.98% similarity)WP_016099737.1 (32.57% identity, 46.55% similarity) US_6555655_B1-13(31.56% identity, 41.15% similarity) US_8461415_B2-29 (30.8% identity,44.42% similarity) APG06560.0 94  95 MTX 96, 97, 98, 99, 100 98, 99, 100APG00427.0 US_2016_0311864_A1-178 (95.61% identity, 97.81% similarity)APG00851.0 US_2016_0311864_A1-222 (89.03% identity, 93.1% similarity)WP_061663532.1 (89.03% identity, 93.1% similarity) APG00585.0US_2016_0366881_A1-116 (82.52% identity, 88.04% similarity)WP_018673409.1 (81.9% identity, 89.57% similarity) APG02701.0 (81.6%identity, 88.34% similarity) WP_001039209.1 (69.94% identity, 81.6%similarity) APG01676.0 (65.45% identity, 77.27% similarity) AOB42285.1(62.73% identity, 75.76% similarity) APG00309.0 US_2016_0355842_A1-56(62.05% identity, 76.81% similarity) APG08411.0 (60.67% identity, 75.91%similarity) APG00989.0 (60.61% identity, 74.85% similarity) APG06730.096  97 Cry 55, 60, 65, 70, 75, 80, 65, 70, 75, 80, 85, APG00288.0US_2016_0311864_A1-122 (51.6% 85, 90, 91, 92, 93, 94, 90, 91, 92, 93,94, identity, 63.2% similarity) 95, 96, 97, 98, 99, 100 95, 96, 97, 98,99, APG00266.0 US_2016_0311864_A1-105 100 (45.86% identity, 56.35%similarity) APG00043.0 US_2016_0304898_A1-69 (26.25% identity, 41.57%similarity) APG01112.0 (25.73% identity, 37.14% similarity) APG01084.0(25.44% identity, 39.14% similarity) APG06801.0 98  99 Bin 50, 55, 60,65, 70, 75, 60, 65, 70, 75, 80, APG05259.0 (54.91% identity, 57.68%similarity) 80, 85, 90, 91, 92, 93, 85, 90, 91, 92, 93, US_8461421 62-6(46.5% identity, 57.0% 94, 95, 96, 97, 98, 99, 94, 95, 96, 97, 98,similarity) 100 99, 100 US_8461421_B2-66 (46.5% identity, 57.0%similarity) US_8461421_B2-148 (39.39% identity, 48.08% similarity)APG06963.0 100 Cry6 55, 60, 65, 70, 75, 80, 75, 80, 85, 90, 91,WP_016110621.1 (88.52% identity, 94.4% 85, 90, 91, 92, 93, 94, 92, 93,94, 95, 96, similarity) 95, 96, 97, 98, 99, 100 97, 98, 99, 100US_8461421_B2-11 (51.93% identity, 72.38% similarity) WP_074651521.1(51.93% identity, 71.27% similarity) WP_016078427.1 (51.93% identity,70.72% similarity) APG00024.0 US_2016_0304898_A1-39 (51.92% identity,70.6% similarity) US_8461421_B2-71 (51.66% identity, 72.38% similarity)WP_000240775.1 (51.66% identity, 71.55% similarity) WP_000240776.1(51.66% identity, 71.55% similarity) WP_061530442.1 (51.66% identity,71.55% similarity) WP_000240777.1 (51.66% identity, 71.27% similarity)SFS89352.1 (51.38% identity, 71.82% similarity) WP_002187778.1 (51.38%identity, 71.55% similarity) APG00631.0 US_2016_0366881_A1-127 (50.83%identity, 71.82% similarity) WP_063549547.1 (50.83% identity, 70.44%similarity) APG00432.0 US_2016_0355842_A1-90 (50.0% identity, 72.38%similarity) APG06971.0 101 102 Cyt 45, 50, 55, 60, 65, 70, 65, 70, 75,80, 85, APG04725.0 (43.51% identity, 62.34% similarity) 75, 80, 85, 90,91, 92, 90, 91, 92, 93, 94, APG01502.0 (42.92% identity, 59.17%similarity) 93, 94, 95, 96, 97, 98, 95, 96, 97, 98, 99, APG00128.0US_2016_0304898_A1-157 99, 100 100 (42.08% identity, 62.5% similarity)WP_016110459.1 (41.67% identity, 62.5% similarity) APG00126.0US_2016_0304898_A1-153 (41.63% identity, 59.18% similarity) APG02923.0(41.25% identity, 60.83% similarity) APG07961.0 (40.91% identity, 60.33%similarity) APG00121.0 US_2016_0311864_A1-29 (40.29% identity, 54.95%similarity) APG08230.0 (39.68% identity, 59.13% similarity) APG08631.0(39.61% identity, 56.47% similarity) APG00177.0 US_2016_0311864_A1-52(39.44% identity, 58.57% similarity) APG00437.0 US_2016_0366881_A1-62(39.22% identity, 56.86% similarity) US_6093695_A-1 (38.02% identity,56.2% similarity) US_6686452-2 (37.65% identity, 55.47% similarity)US_7786351_B2-349 (37.65% identity, 55.47% similarity) US_9121035_B2-18(37.65% identity, 55.47% similarity) APG01246.0 (37.55% identity, 53.88%similarity) APG00468.0 US_2016_0366881_A1-81 (37.36% identity, 53.11%similarity) CA_2267667-2 (37.25% identity, 55.06% similarity) APG00015.0US_2016_0304898_A1-25 (37.0% identity, 53.85% similarity) APG01121.0(36.4% identity, 51.1% similarity) ACI42305.1 (35.18% identity, 55.73%similarity) APG07083.0 103 MTX 5, 10, 15, 20, 25, 30, 5, 10, 15, 20, 25,35, APG08452.0 (35.98% identity, 55.33% similarity) 35, 40, 45, 50, 55,60, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 60, 65, 70, 75, 80, 91, 92,93, 94, 95, 96, 85, 90, 91, 92, 93, 97, 98, 99, 100 94, 95, 96, 97, 98,99, 100 APG07125.0 104 105 Cry 55, 60, 65, 70, 75, 80, 55, 60, 65, 70,75, CA_2844913-203 (50.54% identity, 53.13% 85, 90, 91, 92, 93, 94, 80,85, 90, 91, 92, similarity) 95, 96, 97, 98, 99, 100 93, 94, 95, 96, 97,CA_2844913-204 (50.54% identity, 53.13% 98, 99, 100 similarity)APG07235.0 106 107 MTX 40, 45, 50, 55, 60, 65, 55, 60, 65, 70, 75,APG08330.0 (41.54% identity, 57.21% similarity) 70, 75, 80, 85, 90, 91,80, 85, 90, 91, 92, US_8461415_B2-32 (37.57% identity, 53.24% 92, 93,94, 95, 96, 97, 93, 94, 95, 96, 97, similarity) 98, 99, 100 98, 99, 100WP_001267112.1 (37.3% identity, 52.43% similarity) US_2016_0017363_A1-63(37.1% identity, 51.08% similarity) WP_075718641.1 (36.87% identity,53.58% similarity) US_2016_0017363_A1-59 (36.51% identity, 51.59%similarity) ANS51604.1 (36.51% identity, 51.32% similarity)US_2016_0017363_A1-60 (36.24% identity, 51.06% similarity)US_2016_0017363_A1-61 (35.98% identity, 50.26% similarity)WP_075719153.1 (35.71% identity, 50.0% similarity) WP_043924590.1(35.43% identity, 52.49% similarity) APG07247.0 108 109 Bin 90, 91, 92,93, 94, 95, 94, 95, 96, 97, 98, WP_002187944.1 (91.09% identity, 95.67%96, 97, 98, 99, 100 99, 100 similarity) WP_001258160.1 (88.3% identity,93.89% similarity) CA_2844913-10 (88.07% identity, 93.65% similarity)WP_001258161.1 (88.04% identity, 93.64% similarity) EEL19841.1 (86.77%identity, 91.86% similarity) SCV23895.1 (85.75% identity, 89.31%similarity) APG00118.0 US_2016_0311864_A1-28 (85.52% identity, 87.59%similarity) APG01790.0 (82.94% identity, 87.38% similarity) APG00223.0US_2016_0311864_A1-82 (80.46% identity, 83.91% similarity)WP_002114997.1 (79.9% identity, 88.55% similarity) APG00454.0US_2016_0311864_A1-179 (79.77% identity, 83.68% similarity) APG00335.0US_2016_0366881_A1-18 (79.54% identity, 83.45% similarity) APG00242.0US_2016_0311864_A1-93 (79.54% identity, 83.22% similarity) APG00913.0US_2016_0311864_A1-226 (78.76% identity, 82.3% similarity) EJV43967.1(78.12% identity, 83.21% similarity) WP_070128649.1 (77.86% identity,86.51% similarity) APG00261.0 US_2016_0311864_A1-100 (75.38% identity,85.03% similarity) APG07334.0 110 111 Bin 93, 94, 95, 96, 97, 98, 95,96, 97, 98, 99, APG00419.0 US_2016_0311864_A1-176 99, 100 100 (92.98%identity, 94.74% similarity) APG00229.0 US_2016_0311864_A1-84 (92.57%identity, 94.8% similarity) APG01363.0 (88.86% identity, 92.66%similarity) APG00844.0 US_2016_0311864_A1-221 (87.82% identity, 90.61%similarity) APG00301.0 US_2016_0311864_A1-131 (86.8% identity, 89.59%similarity) APG00353.0 US_2016_0311864_A1-144 (85.79% identity, 88.83%similarity) APG00065.0 US_2016_0177333_A1-31 (85.64% identity, 91.83%similarity) APG00203.0 US_2016_0311864_A1-69 (85.28% identity, 89.09%similarity) APG00412.0 US_2016_0311864_A1-173 (81.64% identity, 85.61%similarity) APG00300.0 US_2016_0355842_A1-53 (80.5% identity, 89.5%similarity) APG09977.0 (80.25% identity, 86.58% similarity) APG07383.0112 113 Cry 96, 97, 98, 99, 100 98, 99, 100 APG00094.0US_2016_0177333_A1-50 (95.79% identity, 97.74% similarity)WP_064532221.1 (92.47% identity, 94.28% similarity) WP_063549999.1(92.17% identity, 94.13% similarity) APG02438.0 (85.57% identity, 90.03%similarity) WP_074651312.1 (58.66% identity, 71.32% similarity)APG00062.0 US_2016_0177333_A1-29 (57.35% identity, 70.74% similarity)US_8759619_B2-24 (35.89% identity, 51.4% similarity) US_8318900_B2-169(35.0% identity, 50.97% similarity) US_8461421_B2-184 (34.92% identity,52.23% similarity) APG06630.0 (33.15% identity, 49.24% similarity)APG00897.0 US_2016_0355842_A1-186 (33.15% identity, 48.27% similarity)APG01199.0 (33.06% identity, 48.58% similarity) WP_002187556.1 (32.67%identity, 46.74% similarity) APG00130.0 US_2016_0177333_A1-72 (32.59%identity, 50.35% similarity) APG00511.0 US_2016_0177333_A1-127 (32.45%identity, 50.35% similarity) US_8759619_B2-26 (32.13% identity, 49.65%similarity) US_8461421_B2-204 (32.08% identity, 46.23% similarity)APG00581.0 US_2016_0355842_A1-133 (31.1% identity, 48.39% similarity)APG00145.0 US_2016_0304898_A1-163 (30.72% identity, 45.09% similarity)WO_2015_118207-2 (30.47% identity, 42.12% similarity) APG00041.0US_2016_0304898_A1-64 (30.45% identity, 45.14% similarity) APG07491.0114 115 Bin 80, 85, 90, 91, 92, 93, 90, 91, 92, 93, 94, SDZ44649.1(93.45% identity, 95.21% similarity) 94, 95, 96, 97, 98, 99, 95, 96, 97,98, 99, APG00988.0 (78.95% identity, 85.46% similarity) 100 100APG00412.0 US_2016_0311864_A1-173 (72.11% identity, 80.9% similarity)WP_002187944.1 (70.97% identity, 81.64% similarity) APG00648.0 (69.83%identity, 77.62% similarity) APG07247.0 (69.76% identity, 79.27%similarity) WP_002114997.1 (69.23% identity, 78.41% similarity)CA_2844913-10 (69.14% identity, 80.25% similarity) APG00261.0US_2016_0311864_A1-100 (69.06% identity, 77.97% similarity)WP_001258160.1 (68.89% identity, 80.0% similarity) WP_001258161.1(68.89% identity, 80.0% similarity) APG00243.0 US_2016_0311864_A1-94(68.59% identity, 77.64% similarity) APG09977.0 (68.59% identity, 76.88%similarity) APG00301.0 US_2016_0311864_A1-131 (68.17% identity, 76.94%similarity) APG00353.0 US_2016_0311864_A1-144 (68.17% identity, 76.94%similarity) APG00203.0 US_2016_0311864_A1-69 (68.17% identity, 76.69%similarity) APG01790.0 (68.07% identity, 78.32% similarity)WP_070128649.1 (67.74% identity, 76.67% similarity) EEL19841.1 (67.65%identity, 78.27% similarity) APG02572.0 (67.31% identity, 75.3%similarity) APG00118.0 US_2016_0311864_A1-28 (66.97% identity, 76.61%similarity) APG00331.0 US_2016_0366881_A1-15 (66.75% identity, 75.68%similarity) APG00242.0 US_2016_0311864_A1-93 (66.74% identity, 75.46%similarity) APG00213.0 US_2016_0311864_A1-76 (66.42% identity, 76.69%similarity) APG00454.0 US_2016_0311864_A1-179 (66.28% identity, 75.46%similarity) APG00223.0 US_2016_0311864_A1-82 (66.28% identity, 75.23%similarity) APG00844.0 US_2016_0311864_A1-221 (66.25% identity, 76.83%similarity) WP_000839920.1 (65.84% identity, 75.5% similarity)APG00335.0 US_2016_0366881_A1-18 (65.83% identity, 75.46% similarity)CA_2844913-1 (65.16% identity, 72.93% similarity) APG07526.0 116 117Vip4 85, 90, 91, 92, 93, 94, 91, 92, 93, 94, 95, APG04931.0 (83.73%identity, 90.05% similarity) 95, 96, 97, 98, 99, 100 96, 97, 98, 99, 100SCL96146.1 (62.4% identity, 75.1% similarity) WP_053512697.1 (62.4%identity, 75.0% similarity) WP_048561796.1 (62.2% identity, 75.2%similarity) US_2016_0339078_A1-28124 (61.04% identity, 70.56%similarity) SCC38903.1 (59.9% identity, 70.84% similarity) APG09545.0(59.09% identity, 70.75% similarity) US_2016_0339078_A1-29674 (58.97%identity, 73.45% similarity) APG00106.0 US_2016_0304898_A1-134 (56.28%identity, 70.28% similarity) APG09984.0 (56.26% identity, 70.19%similarity) APG07606.0 118 120, 119 MTX 90, 91, 92, 93, 94, 95, 90, 91,92, 93, 94, APG09460.0 (86.96% identity, 88.04% similarity) 96, 97, 98,99, 100 95, 96, 97, 98, 99, APG00416.0 US_2016 0355842_A1-82 (86.41% 100identity, 87.5% similarity) WP_072335847.1 (45.58% identity, 60.05%similarity) APG00793.0 US_2016_0355842_A1-170 (35.62% identity, 54.35%similarity) SDZ23421.1 (32.04% identity, 46.25% similarity)US_8461415_B2-36 (31.13% identity, 44.85% similarity) US_8829279_B2-11(30.12% identity, 47.16% similarity) APG00451.0 US_2016_0355842_A1-94(29.38% identity, 46.13% similarity) APG00051.0 US_2016_0304898_A1-81(28.41% identity, 42.96% similarity) APG06242.0 (28.23% identity, 42.82%similarity) WP_075716873.1 (27.61% identity, 41.04% similarity)APG00594.0 US_2016_0355842_A1-137 (27.6% identity, 39.95% similarity)APG00911.0 US_2016_0355842_A1-189 (27.59% identity, 41.51% similarity)APG00170.0 US_2016_0355842_A1-13 (27.25% identity, 42.03% similarity)APG00293.0 US_2016_0355842_A1-51 (27.11% identity, 41.23% similarity)APG00959.0 US_2016_0355842_A1-194 (27.07% identity, 41.95% similarity)APG05615.0 (27.0% identity, 41.75% similarity) APG00481.0US_2016_0355842_A1-103 (26.89% identity, 43.52% similarity) SCA97082.1(26.75% identity, 41.5% similarity) WP_000844424.1 (26.75% identity,41.5% similarity) WP_000844425.1 (26.75% identity, 41.5% similarity)APG00764.0 US_2016_0355842_A1-168 (26.65% identity, 43.03% similarity)APG08794.0 (26.32% identity, 39.35% similarity) APG05372.0 (26.19%identity, 39.95% similarity) APG00597.0 US_2016_0355842_A1-139 (26.18%identity, 42.39% similarity) APG00854.0 US_2016_0355842_A1-179 (26.17%identity, 39.25% similarity) APG00269.0 US_2016_0355842_A1-46 (26.11%identity, 39.86% similarity) APG09338.0 (26.07% identity, 40.67%similarity) APG00154.0 US_2016_0355842_A1-5 (26.02% identity, 39.28%similarity) APG00322.0 US_2016_0355842_A1-59 (25.8% identity, 41.77%similarity) APG00635.0 US_2016_0311864_A1-201 (25.79% identity, 43.07%similarity) APG00393.0 US_2016_0355842_A1-76 (25.74% identity, 42.08%similarity) APG00646.0 US_2016_0355842_A1-201 (25.71% identity, 40.24%similarity) APG09211.0 (25.6% identity, 38.07% similarity) APG00020.0US_2016_0304898_A1-33 (25.47% identity, 38.92% similarity)US_8461415_B2-37 (25.45% identity, 39.09% similarity) APG00475.0US_2016_0366881_A1-90 (25.41% identity, 38.93% similarity) APG00414.0US_2016_0355842_A1-81 (25.3% identity, 39.86% similarity) APG00268.0US_2016_0355842_A1-45 (25.24% identity, 39.76% similarity) APG00483.0US_2016_0355842_A1-104 (25.18% identity, 38.61% similarity) APG07679.0121 122 Cry 60, 65, 70, 75, 80, 85, 75, 80, 85, 90, 91, CA_2866166-326(59.2% identity, 70.21% 90, 91, 92, 93, 94, 95, 92, 93, 94, 95, 96,similarity) 96, 97, 98, 99, 100 97, 98, 99, 100 JP_2002_335967-21 (59.2%identity, 70.14% similarity) US_8759619_B2-25 (58.88% identity, 68.16%similarity) WO_2016_109214-2 (58.26% identity, 69.92% similarity)US_2014_0096281_A1-4 (57.4% identity, 69.12% similarity) APG00687.0(57.34% identity, 69.23% similarity) APG06650.0 (56.99% identity, 68.75%similarity) APG07889.0 (56.42% identity, 66.22% similarity) APG03498.0(56.04% identity, 65.71% similarity) APG01915.0 (54.25% identity, 65.8%similarity) APG00007.0 US_2016_0304898_A1-11 (54.03% identity, 66.01%similarity) US_8461421_B2-68 (53.32% identity, 66.23% similarity)US_8461421_B2-70 (53.24% identity, 64.46% similarity) US_8461421_B2-91(53.02% identity, 63.13% similarity) APG06253.0 (52.68% identity, 64.93%similarity) US_2016_0122399_A1-4 (52.41% identity, 62.51% similarity)JP_2002_335967-3 (51.82% identity, 61.39% similarity) APG00188.0US_2016_0311864_A1-57 (51.7% identity, 64.32% similarity) APG00974.0(51.33% identity, 62.87% similarity) APG00056.0 US_2016_0311864_A1-1(51.17% identity, 61.68% similarity) US_8318900_B2-82 (51.03% identity,62.15% similarity) APG06739.0 (50.96% identity, 62.65% similarity)US_8759619_B2-23 (50.87% identity, 63.27% similarity) US_8759619_B2-6(50.87% identity, 63.27% similarity) APG00430.0 US_2016_0366881_A1-54(50.77% identity, 60.98% similarity) ADO32760.1 (50.69% identity, 61.95%similarity) APG01329.0 (50.65% identity, 62.48% similarity) APG07803.0(50.54% identity, 63.91% similarity) APG00068.0 US_2016_0177333_A1-35(50.51% identity, 61.34% similarity) US_8318900_B2-38 (50.47% identity,62.49% similarity) US_8318900_B2-87 (50.47% identity, 62.49% similarity)APG00710.0 (50.4% identity, 62.56% similarity) US_8461421_B2-98 (50.29%identity, 61.11% similarity) US_8461421_B2-98 (50.29% identity, 61.11%similarity) ADO32759.1 (50.04% identity, 61.73% similarity) AAG36711.1(50.04% identity, 61.66% similarity) APG07799.0 123 124 Cry 45, 50, 55,60, 65, 70, 60, 65, 70, 75, 80, US_8318900_B2-111 (41.63% identity,57.38% 75, 80, 85, 90, 91, 92, 85, 90, 91, 92, 93, similarity) 93, 94,95, 96, 97, 98, 94, 95, 96, 97, 98, US_8318900_B2-80 (41.34% identity,56.98% 99, 100 99, 100 similarity) WP_016099738.1 (40.52% identity,57.14% similarity) US_8461415_B2-28 (34.77% identity, 49.46% similarity)US_8461415_B2-1 (33.69% identity, 48.25% similarity) US_8461421_B2-120(29.55% identity, 42.48% similarity) US_8318900_B2-83 (29.45% identity,47.93% similarity) ADD71694.1 (29.05% identity, 47.26% similarity)APG00864.0 (28.86% identity, 45.61% similarity) CA_2516349-9 (28.84%identity, 41.58% similarity) APG00664.0 (28.68% identity, 42.89%similarity) APG00981.0 (28.41% identity, 45.71% similarity) ADM64568.1(28.4% identity, 42.12% similarity) APG00005.0 US_2016_0177333_A1-4(28.28% identity, 41.9% similarity) APG00076.0 US_2016_0177333_A1-41(28.23% identity, 44.42% similarity) APG00726.0 (28.21% identity, 42.0%similarity) CA_2516349-7 (28.21% identity, 40.42% similarity) APG00133.0US_2016_0304898_A1-160 (28.15% identity, 45.33% similarity) APG00972.0(27.99% identity, 40.55% similarity) APG00785.0 (27.74% identity, 43.77%similarity) WP_050845813.1 (27.71% identity, 41.89% similarity)ADV57670.1 (27.71% identity, 41.76% similarity) BAB72016.2 (27.47%identity, 42.9% similarity) APG00565.0 (27.38% identity, 42.93%similarity) CA_2516349-49 (27.33% identity, 42.63% similarity)APG00003.0 US_2016_0304898_A1-4 (27.14% identity, 42.11% similarity)CA_2426793-8 (27.04% identity, 44.43% similarity) APG00149.0US_2016_0304898_A1-172 (27.02% identity, 44.24% similarity) APG08430.0(27.01% identity, 42.78% similarity) CA_2426793-7 (27.0% identity,44.37% similarity) CA_2426793-18 (26.82% identity, 44.07% similarity)APG00169.0 US_2016_0304898_A1-181 (26.72% identity, 44.44% similarity)APG00040.0 US_2016_0304898_A1-62 (26.44% identity, 43.66% similarity)APG07954.0 (26.41% identity, 42.18% similarity) APG00802.0 (26.38%identity, 40.08% similarity) APG00582.0 US_2016_0366881_A1-112 (26.37%identity, 43.34% similarity) EP_2458009-20 (26.36% identity, 44.37%similarity) APG01992.0 (26.33% identity, 38.81% similarity) APG00152.0US_2016_0311864_A1-36 (26.28% identity, 43.36% similarity) APG01844.0(26.27% identity, 43.82% similarity) APG00965.0 (26.26% identity, 43.5%similarity) CA_2426793-5 (26.21% identity, 44.49% similarity) APG00643.0(26.18% identity, 44.21% similarity) CA_2426793-10 (26.17% identity,44.43% similarity) APG00088.0 US_2016_0304898_A1-116 (26.17% identity,40.72% similarity) APG00986.0 (26.16% identity, 40.34% similarity)APG00800.0 US_2016_0355842_A1-172 (26.15% identity, 44.07% similarity)APG00125.0 US_2016_0304898_A1-151 (26.06% identity, 42.43% similarity)APG00537.0 (25.94% identity, 39.95% similarity) APG00581.0US_2016_0355842_A1-133 (25.88% identity, 40.4% similarity) APG09877.0(25.81% identity, 43.23% similarity) OMH24296.1 (25.75% identity, 43.92%similarity) APG00555.0 (25.75% identity, 42.65% similarity) APG00389.0US_2016_0366881_A1-46 (25.68% identity, 43.48% similarity) APG00677.0(25.68% identity, 41.67% similarity) APG06528.0 (25.48% identity, 41.57%similarity) APG00066.0 US_2016_0177333_A1-33 (25.48% identity, 40.25%similarity) APG06630.0 (25.42% identity, 39.56% similarity) APG00057.0US_2016_0304898_A1-88 (25.32% identity, 40.39% similarity) APG00004.0US_2016_0304898_A1-7 (25.3% identity, 41.59% similarity) APG05810.0(25.1% identity, 41.95% similarity) APG00343.0 (25.03% identity, 42.02%similarity) APG07868.0 125 Bin 96, 97, 98, 99, 100 98, 99, 100APG08195.0 (96.07% identity, 97.54% similarity) APG00178.0US_2016_0311864_A1-54 (95.33% identity, 97.3% similarity) APG00727.0US_2016_0311864_A1-210 (91.15% identity, 93.37% similarity) APG00315.0US_2016_0311864_A1-137 (88.7% identity, 91.65% similarity) APG00449.0US_2016_0366881_A1-71 (29.37% identity, 44.06% similarity) APG00532.0US_2016_0311864_A1-189 (28.97% identity, 43.46% similarity) APG00880.0US_2016_0366881_A1-134 (28.61% identity, 46.34% similarity) APG00191.0US_2016_0304898_A1-191 (28.27% identity, 45.13% similarity) APG00649.0(28.08% identity, 45.21% similarity) APG00215.0 US_2016_0311864_A1-78(28.07% identity, 42.92% similarity) APG00380.0 US_2016_0366881_A1-43(27.65% identity, 44.7% similarity) APG03147.0 (27.44% identity, 44.19%similarity) WP_048517129.1 (27.17% identity, 44.73% similarity)APG00729.0 (27.08% identity, 37.71% similarity) APG09768.0 (26.98%identity, 41.54% similarity) CA_2866166-398 (26.97% identity, 43.98%similarity) US_9321814_B2-2 (26.83% identity, 41.69% similarity)US_9321814_B2-3 (26.83% identity, 41.69% similarity) APG00669.0US_2016_0355842_A1-156 (26.74% identity, 41.8% similarity) APG00386.0US_2016_0311864_A1-158 (26.73% identity, 42.4% similarity) CA_2844913-49(26.68% identity, 42.38% similarity) APG00090.0 US_2016_0177333_A1-49(26.67% identity, 44.37% similarity) APG00837.0 (26.59% identity, 45.41%similarity) CA_2866166-394 (26.46% identity, 41.41% similarity)APG00494.0 (26.44% identity, 41.78% similarity) APG00050.0US_2016_0304898_A1-80 (26.37% identity, 42.2% similarity)US_9321814_B2-5 (26.29% identity, 41.35% similarity) APG00157.0US_2016_0355842_A1-7 (26.22% identity, 42.0% similarity) APG00230.0US_2016_0311864_A1-85 (26.2% identity, 42.6% similarity) US_7692068_B2-2(26.19% identity, 40.48% similarity) APG00095.0 US_2016_0177333_A1-52(26.17% identity, 41.16% similarity) APG06401.0 (26.16% identity, 40.85%similarity) US_9321814_B2-4 (26.16% identity, 40.35% similarity)APG00706.0 (26.14% identity, 40.2% similarity) APG08005.0 (26.09%identity, 41.09% similarity) APG00602.0 US_2016_0366881_A1-118 (26.08%identity, 43.31% similarity) US_7692068_B2-3 (26.08% identity, 40.09%similarity) APG00231.0 US_2016_0311864_A1-86 (26.05% identity, 41.06%similarity) APG06051.0 (25.98% identity, 37.7% similarity) APG00700.0US_2016_0311864_A1-207 (25.95% identity, 40.72% similarity) APG00063.0US_2016_0304898_A1-95 (25.89% identity, 41.26% similarity) APG00568.0US_2016_0311864_A1-391 (25.83% identity, 40.62% similarity) APG00377.0US_2016_0311864_A1-154 (25.77% identity, 40.75% similarity) APG00373.0US_2016_0311864_A1-151 (25.75% identity, 43.68% similarity) ABI15165.1(25.68% identity, 42.27% similarity) APG00629.0 US_2016_0366881_A1-126(25.66% identity, 40.93% similarity) APG00642.0 US_2016_0311864_A1-202(25.64% identity, 39.95% similarity) US_5827684_A-5.01 (25.45% identity,42.05% similarity) WP_036166959.1 (25.45% identity, 41.59% similarity)APG00557.0 (25.43% identity, 38.48% similarity) APG00257.0US_2016_0355842_A1-42 (25.4% identity, 43.94% similarity) APG00287.0US_2016_0311864_A1-121 (25.39% identity, 40.84% similarity) APG00356.0US_2016_0311864_A1-147 (25.39% identity, 40.84% similarity)US_5827684_A-22.01 (25.37% identity, 39.66% similarity) APG00413.0US_2016_0311864_A1-174 (25.33% identity, 41.19% similarity) APG00474.0US_2016_0366881_A1-89 (25.28% identity, 39.05% similarity) CAA04291.1(25.23% identity, 41.82% similarity) US_5827684_A-7.01 (25.23% identity,41.82% similarity) WP_036216620.1 (25.23% identity, 41.59% similarity)5G37_A (25.23% identity, 41.36% similarity) APG00075.0US_2016_0177333_A1-40 (25.19% identity, 41.09% similarity) APG00459.0US_2016_0311864_A1-181 (25.16% identity, 38.06% similarity) APG07889.0126 127 Cry 80, 85, 90, 91, 92, 93, 85, 90, 91, 92, 93, US_8461421_B2-70(77.99% identity, 84.42% 94, 95, 96, 97, 98, 99, 94, 95, 96, 97, 98,similarity) 100 99, 100 APG03519.0 (72.35% identity, 80.3% similarity)CA_2866166-327 (71.2% identity, 79.04% similarity) JP_2002_335967-2(69.58% identity, 77.24% similarity) APG05311.0 (69.01% identity, 76.83%similarity) APG03498.0 (57.67% identity, 68.17% similarity)US_8461421_B2-91 (57.09% identity, 67.89% similarity) US_8461421_B2-91(57.09% identity, 67.89% similarity) US_8759619_B2-25 (56.66% identity,66.82% similarity) APG07679.0 (56.42% identity, 66.22% similarity)APG00430.0 US_2016_0366881_A1-54 (56.25% identity, 66.9% similarity)APG00974.0 (55.53% identity, 66.28% similarity) APG00974.0 (55.53%identity, 66.28% similarity) APG08005.0 128 129 Bin 90, 91, 92, 93, 94,95, 95, 96, 97, 98, 99, APG08361.0 (90.38% identity, 93.92% similarity)96, 97, 98, 99, 100 100 APG00757.0 US_2016_0311864_A1-215 (89.37%identity, 94.18% similarity) APG00482.0 US_2016_0366881_A1-94 (88.86%identity, 93.67% similarity) APG00741.0 US_2016_0311864_A1-213 (82.53%identity, 87.59% similarity) WP_017154552.1 (65.92% identity, 75.62%similarity) APG03279.0 (64.93% identity, 73.38% similarity) APG00596.0US_2016_0311864_A1-196 (63.46% identity, 74.32% similarity) APG00413.0US_2016_0311864_A1-174 (62.59% identity, 72.57% similarity)US_9321814_B2-2 (61.73% identity, 71.36% similarity) US_9321814_B2-3(61.73% identity, 71.11% similarity) SFQ92148.1 (61.52% identity, 65.32%similarity) WP_050001305.1 (61.48% identity, 71.6% similarity)APG00230.0 US_2016_0311864_A1-85 (61.12% identity, 71.88% similarity)APG00060.0 US_2016_0304898_A1-90 (61.01% identity, 64.81% similarity)APG00090.0 US_2016_0177333_A1-49 (60.5% identity, 70.75% similarity)US_9321814_B2-4 (60.25% identity, 69.63% similarity) APG08195.0 130 Bin96, 97, 98, 99, 100 98, 99, 100 APG07868.0 (96.07% identity, 97.54%similarity) APG00178.0 US_2016_0311864_A1-54 (95.58% identity, 97.54%similarity) APG00727.0 US_2016_0311864_A1-210 (91.65% identity, 93.86%similarity) APG00315.0 US_2016_0311864_A1-137 (88.94% identity, 93.12%similarity) APG00729.0 (30.07% identity, 42.32% similarity) APG00449.0US_2016_0366881_A1-71 (29.84% identity, 45.69% similarity) APG00191.0US_2016_0304898_A1-191 (28.47% identity, 45.65% similarity) APG00532.0US_2016_0311864_A1-189 (28.41% identity, 44.57% similarity) APG00649.0(28.31% identity, 45.21% similarity) APG00380.0 US_2016_0366881_A1-43(28.21% identity, 46.33% similarity) CA_2866166-398 (28.02% identity,44.79% similarity) CA_2866166-394 (27.35% identity, 43.47% similarity)APG00880.0 US_2016_0366881_A1-134 (27.19% identity, 46.81% similarity)APG00090.0 US_2016_0177333_A1-49 (27.13% identity, 44.6% similarity)APG00230.0 US_2016_0311864_A1-85 (27.11% identity, 43.51% similarity)APG00386.0 US_2016_0311864_A1-158 (26.98% identity, 42.56% similarity)APG06401.0 (26.96% identity, 41.65% similarity) WP_036166959.1 (26.93%identity, 44.03% similarity) APG00215.0 US_2016_0311864_A1-78 (26.93%identity, 42.86% similarity) APG00157.0 US_2016_0355842_A1-7 (26.77%identity, 42.26% similarity) APG00494.0 (26.77% identity, 41.37%similarity) APG00050.0 US_2016_0304898_A1-80 (26.71% identity, 43.27%similarity) APG09768.0 (26.71% identity, 41.67% similarity) CAA04291.1(26.7% identity, 44.26% similarity) WP_036216620.1 (26.7% identity,44.03% similarity) US_9321814_B2-2 (26.67% identity, 42.0% similarity)US_9321814_B2-3 (26.67% identity, 42.0% similarity) APG00669.0US_2016_0355842_A1-156 (26.67% identity, 41.56% similarity) ABI15165.1(26.62% identity, 43.06% similarity) APG00629.0 US_2016_0366881_A1-126(26.55% identity, 41.37% similarity) APG08005.0 (26.52% identity, 41.96%similarity) US_7692068_B2-2 (26.49% identity, 41.43% similarity)APG00063.0 US_2016_0304898_A1-95 (26.47% identity, 42.44% similarity)US_8318900_B2-72 (26.46% identity, 40.58% similarity) US_5827684_A-5.01(26.39% identity, 43.06% similarity) APG00231.0 US_2016_0311864_A1-86(26.37% identity, 41.32% similarity) APG00706.0 (26.34% identity, 41.39%similarity) US_8829279_B2-39 (26.29% identity, 43.43% similarity)APG03147.0 (26.23% identity, 42.38% similarity) US_5827684_A-7.01(26.16% identity, 42.82% similarity) 5G37_A (26.16% identity, 42.36%similarity) APG00568.0 US_2016_0311864_A1-391 (26.15% identity, 40.66%similarity) US_9321814_B2-5 (26.13% identity, 41.67% similarity)APG00095.0 US_2016_0177333_A1-52 (26.12% identity, 41.74% similarity)US_7692068_B2-3 (26.11% identity, 41.05% similarity) APG00602.0US_2016_0366881_A1-118 (26.02% identity, 43.44% similarity)US_9321814_B2-4 (26.0% identity, 40.67% similarity) APG00837.0 (25.93%identity, 46.03% similarity) P12963.1 (25.93% identity, 42.36%similarity) APG00700.0 US_2016_0311864_A1-207 (25.89% identity, 41.29%similarity) APG00413.0 US_2016_0311864_A1-174 (25.82% identity, 41.58%similarity) APG00373.0 US_2016_0311864_A1-151 (25.75% identity, 43.45%similarity) APG00377.0 US_2016_0311864_A1-154 (25.71% identity, 41.1%similarity) APG00287.0 US_2016_0311864_A1-121 (25.71% identity, 40.88%similarity) APG00356.0 US_2016_0311864_A1-147 (25.71% identity, 40.88%similarity) APG00557.0 (25.65% identity, 38.48% similarity) APG06051.0(25.58% identity, 38.25% similarity) CA_2844913-49 (25.56% identity,41.48% similarity) CAA73757.1 (25.52% identity, 41.53% similarity)APG00459.0 US_2016_0311864_A1-181 (25.48% identity, 38.22% similarity)APG00642.0 US_2016_0311864_A1-202 (25.17% identity, 39.26% similarity)US_5827684_A-22.01 (25.15% identity, 39.38% similarity) APG00035.0US_2016_0304898_A1-58 (25.11% identity, 41.48% similarity) APG00735.0(25.05% identity, 41.1% similarity) APG00731.0 US_2016_0311864_A1-211(25.05% identity, 40.44% similarity) APG08330.0 131 132 MTX 35, 40, 45,50, 55, 60, 55, 60, 65, 70, 75, APG07235.0 (41.54% identity, 57.21%similarity) 65, 70, 75, 80, 85, 90, 80, 85, 90, 91, 92, US_8461415_B2-32(33.78% identity, 48.53% 91, 92, 93, 94, 95, 96, 93, 94, 95, 96, 97,similarity) 97, 98, 99, 100 98, 99, 100 WP_001267112.1 (33.78% identity,47.99% similarity) AGC39300.1 (32.67% identity, 48.63% similarity)US_2016_0017363_A1-59 (32.64% identity, 50.13% similarity) ANS51604.1(32.64% identity, 49.87% similarity) WP_075718641.1 (32.2% identity,48.43% similarity) US_2016_0017363_A1-60 (32.11% identity, 49.61%similarity) US_2016_0017363_A1-61 (31.85% identity, 48.83% similarity)WP_023524027.1 (31.78% identity, 50.39% similarity) WP_043924590.1(31.63% identity, 47.45% similarity) WP_075719153.1 (30.77% identity,45.41% similarity) WP_075716686.1 (30.77% identity, 45.16% similarity)APG08361.0 133 134 Bin 95, 96, 97, 98, 99, 100 98, 99, 100 APG00757.0US_2016_0311864_A1-215 (94.94% identity, 97.97% similarity) APG00482.0US_2016_0366881_A1-94 (94.68% identity, 97.72% similarity) APG08005.0(90.38% identity, 93.92% similarity) APG00741.0 US_2016_0311864_A1-213(88.35% identity, 91.65% similarity) WP_017154552.1 (67.08% identity,76.73% similarity) SFQ92148.1 (66.58% identity, 69.11% similarity)APG00060.0 US_2016_0304898_A1-90 (66.08% identity, 68.86% similarity)APG00596.0 US_2016_0311864_A1-196 (65.68% identity, 75.56% similarity)APG03279.0 (64.84% identity, 74.31% similarity) APG00413.0US_2016_0311864_A1-174 (63.59% identity, 73.57% similarity)WP_050001305.1 (62.22% identity, 71.36% similarity) APG00230.0US_2016_0311864_A1-85 (61.12% identity, 72.62% similarity)US_9321814_B2-2 (60.54% identity, 71.81% similarity) US_9321814_B2-3(60.54% identity, 71.57% similarity) APG08369.0 135 136 MTX 65, 70, 75,80, 85, 90, 80, 85, 90, 91, 92, WP_061885189.1 (69.6% identity, 78.42%91, 92, 93, 94, 95, 96, 93, 94, 95, 96, 97, similarity) 97, 98, 99, 10098, 99, 100 APG00552.0 US_2016_0366881_A1-110 (64.46% identity, 77.41%similarity) APG00541.0 US_2016_0355842_A1-119 (57.81% identity, 76.25%similarity) APG00244.0 US_2016_0355842_A1-33 (57.81% identity, 75.31%similarity) WP_016078640.1 (57.75% identity, 73.56% similarity)APG00509.0 US_2016_0355842_A1-113 (57.19% identity, 75.62% similarity)APG00896.0 US_2016_0355842_A1-185 (55.31% identity, 70.0% similarity)WP_006284936.1 (53.78% identity, 69.49% similarity) WP_065212006.1(51.9% identity, 68.51% similarity) APG00633.0 US_2016_0355842_A1-148(51.59% identity, 66.86% similarity) WP_036654376.1 (51.38% identity,65.44% similarity) WP_033699741.1 (50.14% identity, 64.93% similarity)APG00362.0 US_2016_0366881_A1-33 (50.0% identity, 70.55% similarity)APG08430.0 137 Cry 45, 50, 55, 60, 65, 70, 65, 70, 75, 80, 85,APG00133.0 US_2016_0304898_A1-160 75, 80, 85, 90, 91, 92, 90, 91, 92,93, 94, (43.25% identity, 60.2% similarity) 93, 94, 95, 96, 97, 98, 95,96, 97, 98, 99, APG00582.0 US_2016_0366881_A1-112 99, 100 100 (42.56%identity, 55.77% similarity) APG00125.0 US_2016_0304898_A1-151 (39.25%identity, 54.98% similarity) APG00040.0 US_2016_0304898_A1-62 (38.85%identity, 56.1% similarity) APG00028.0 US_2016_0304898_A1-47 (36.66%identity, 51.16% similarity) APG00389.0 US_2016_0366881_A1-46 (36.63%identity, 49.93% similarity) APG00169.0 US_2016_0304898_A1-181 (35.74%identity, 52.25% similarity) US_8461421_B2-240 (33.57% identity, 47.71%similarity) US_8318900_B2-101 (33.33% identity, 49.32% similarity)US_8318900_B2-56 (33.2% identity, 49.12% similarity) US_8318900_B2-7(33.2% identity, 49.12% similarity) APG00606.0 (33.11% identity, 45.91%similarity) US_8318900_B2-147 (33.02% identity, 49.4% similarity)CA_2314429-50 (32.96% identity, 50.07% similarity) CA_2314429-55 (32.96%identity, 49.93% similarity) AJW76684.1 (32.93% identity, 47.04%similarity) CA_2314429-45 (32.81% identity, 49.93% similarity)APG00643.0 (32.64% identity, 47.66% similarity) CA_2314429-27 (32.53%identity, 50.21% similarity) APG00965.0 (32.43% identity, 48.38%similarity) APG07954.0 (32.26% identity, 46.91% similarity) APG00800.0US_2016_0355842_A1-172 (32.09% identity, 48.07% similarity) APG00537.0(32.04% identity, 45.13% similarity) APG00164.0 US_2016_0311864_A1-41(31.98% identity, 46.0% similarity) APG00084.0 US_2016_0311864_A1-15(31.92% identity, 43.97% similarity) APG00613.0 US_2016_0355842_A1-142(31.81% identity, 46.75% similarity) APG00785.0 (31.72% identity, 48.39%similarity) ADM64568.1 (31.69% identity, 45.38% similarity) APG06528.0(31.54% identity, 46.42% similarity) APG06921.0 (31.5% identity, 44.98%similarity) APG00802.0 (31.41% identity, 44.37% similarity) APG00003.0US_2016_0304898_A1-4 (31.4% identity, 46.25% similarity) APG00152.0US_2016_0311864_A1-36 (31.22% identity, 44.44% similarity) APG00514.0(31.0% identity, 46.23% similarity) APG00081.0 US_2016_0304898_A1-105(30.72% identity, 48.17% similarity) APG05660.0 (30.67% identity, 45.67%similarity) APG00677.0 (30.56% identity, 47.18% similarity) APG00116.0US_2016_0311864_A1-25 (30.43% identity, 45.52% similarity) APG00864.0(30.4% identity, 44.93% similarity) APG00004.0 US_2016_0304898_A1-7(30.37% identity, 46.05% similarity) KIQ78153.1 (30.37% identity, 46.02%similarity) APG09376.0 (30.03% identity, 45.38% similarity) APG08452.0138 MTX 5, 10, 15, 20, 25, 30, 5, 10, 15, 20, 25, 30, APG07083.0 (35.98%identity, 55.33% similarity) 35, 40, 45, 50, 55, 60, 35, 40, 45, 50, 55,65, 70, 75, 80, 85, 90, 60, 65, 70, 75, 80, 91, 92, 93, 94, 95, 96, 85,90, 91, 92, 93, 97, 98, 99, 100 94, 95, 96, 97, 98, 99, 100 APG08835.0139 140 Cry 50, 55, 60, 65, 70, 75, 60, 65, 70, 75, 80, APG00217.0US_2016_0355842_A1-24 (45.09% 80, 85, 90, 91, 92, 93, 85, 90, 91, 92,93, identity, 55.75% similarity) 94, 95, 96, 97, 98, 99, 94, 95, 96, 97,98, APG00217.0 US_2016_0355842_A1-24 (45.09% 100 99, 100 identity,55.75% similarity) APG00622.0 (35.88% identity, 47.19% similarity)APG00622.0 (35.88% identity, 47.19% similarity) APG00663.0 (32.66%identity, 44.78% similarity) APG00663.0 (32.66% identity, 44.78%similarity) CA_2595901-12 (32.61% identity, 43.04% similarity)CA_2595901-12 (32.61% identity, 43.04% similarity) CA_2866166-433(32.55% identity, 42.98% similarity) CA_2866166-433 (32.55% identity,42.98% similarity) CA_2866166-434 (32.55% identity, 42.98% similarity)CA_2866166-434 (32.55% identity, 42.98% similarity) CA_2595901-15 (31.2%identity, 44.21% similarity) CA_2595901-15 (31.2% identity, 44.21%similarity) US_8461415_B2-34 (30.72% identity, 43.24% similarity)US_8461415_B2-34 (30.72% identity, 43.24% similarity) US_7923602_B2-4(30.15% identity, 41.05% similarity) US_7923602_B2-4 (30.15% identity,41.05% similarity) US_8318900_B2-68 (30.13% identity, 41.7% similarity)US_8318900_B2-68 (30.13% identity, 41.7% similarity) US_8318900_B2-68(30.13% identity, 41.7% similarity) US_2011_0214208_A1-9 (30.11%identity, 39.78% similarity) US_2011_0214208_A1-9 (30.11% identity,39.78% similarity) APG08992.0 141 142 Vip4 50, 55, 60, 65, 70, 75, 65,70, 75, 80, 85, APG04069.0 (51.09% identity, 65.84% similarity) 80, 85,90, 91, 92, 93, 90, 91, 92, 93, 94, WP_016123960.1 (50.46% identity,67.52% 94, 95, 96, 97, 98, 99, 95, 96, 97, 98, 99, similarity) 100 100AGT29561.1 (48.35% identity, 63.04% similarity) APG07526.0 (46.98%identity, 63.81% similarity) APG04931.0 (45.95% identity, 63.06%similarity) CA_2844913-129 (44.89% identity, 60.5% similarity)CA_2844913-130 (44.89% identity, 60.5% similarity) SCC34829.1 (44.84%identity, 59.98% similarity) APG01474.0 (44.79% identity, 61.77%similarity) US_2016_0339078_A1-29674 (44.5% identity, 59.88% similarity)APG09984.0 (44.48% identity, 59.84% similarity) APG00106.0US_2016_0304898_A1-134 (44.31% identity, 58.91% similarity)US_2016_0339078_A1-28464 (43.11% identity, 57.09% similarity) APG01927.0(42.97% identity, 58.51% similarity) APG07790.0 (41.91% identity, 58.09%similarity) WP_048561796.1 (41.83% identity, 59.45% similarity)SCL96146.1 (41.83% identity, 59.25% similarity) WP_053512697.1 (41.83%identity, 59.15% similarity) SCC38903.1 (41.71% identity, 59.36%similarity) US_2016_0339078_A1-28124 (40.14% identity, 55.37%similarity) APG09100.0 143 144 MTX 50, 55, 60, 65, 70, 75, 65, 70, 75,80, 85, APG04586.0 (49.01% identity, 63.66% similarity) 80, 85, 90, 91,92, 93, 90, 91, 92, 93, 94, US_8318900 82-77 (48.19% identity, 62.95%94, 95, 96, 97, 98, 99, 95, 96, 97, 98, 99, similarity) 100 100APG00645.0 US_2016_0355842_A1-151 (36.29% identity, 53.19% similarity)APG00860.0 US_2016_0355842_A1-180 (36.29% identity, 52.91% similarity)APG00718.0 (36.21% identity, 50.14% similarity) APG00425.0US_2016_0355842_A1-87 (34.85% identity, 51.47% similarity) APG03324.0(33.61% identity, 53.28% similarity) APG00586.0 US_2016_0355842_A1-135(33.51% identity, 52.86% similarity) APG00420.0 US_2016_0355842_A1-85(33.42% identity, 52.99% similarity) APG00323.0 US_2016_0355842_A1-60(32.97% identity, 52.43% similarity) APG03101.0 (31.69% identity, 48.63%similarity) APG09155.0 145 146 MTX 95, 96, 97, 98, 99, 100 98, 99, 100ANN35739.1 (94.95% identity, 97.48% similarity) CA_2844913-102 (94.32%identity, 97.48% similarity) ANN35530.1 (82.19% identity, 89.38%similarity) APG00423.0 US_2016_0366881_A1-51 (78.12% identity, 85.0%similarity) US_2015_0218583_A1-5 (64.04% identity, 78.65% similarity)US_2015_0218583_A1-6 (62.77% identity, 76.0% similarity) APG00146.0US_2016_0304898_A1-166 (61.49% identity, 76.4% similarity)WP_000794514.1 (60.8% identity, 75.31% similarity) APG00590.0US_2016_0311864_A1-193 (58.63% identity, 71.13% similarity) APG00387.0US_2016_0366881_A1-45 (58.23% identity, 74.7% similarity) APG06228.0(57.98% identity, 73.01% similarity) APG09055.0 (56.51% identity, 73.08%similarity) APG00980.0 (56.36% identity, 68.5% similarity)US_8318900_B2-78 (55.96% identity, 68.81% similarity) APG09492.0 (55.88%identity, 71.18% similarity) APG00563.0 US_2016_0355842_A1-127 (55.49%identity, 70.92% similarity) APG00794.0 US_2016_0355842_A1-171 (55.33%identity, 70.41% similarity) APG00938.0 US_2016_0355842_A1-190 (55.19%identity, 71.51% similarity) APG00351.0 US_2016_0366881_A1-28 (55.17%identity, 67.82% similarity) APG09211.0 147 148 MTX 60, 65, 70, 75, 80,85, 75, 80, 85, 90, 91, APG00170.0 US_2016_0355842_A1-13 (59.76% 90, 91,92, 93, 94, 95, 92, 93, 94, 95, 96, identity, 68.81% similarity) 96, 97,98, 99, 100 97, 98, 99, 100 APG00293.0 US_2016_0355842_A1-51 (56.46%identity, 66.51% similarity) APG00051.0 US_2016_0304898_A1-81 (55.61%identity, 71.36% similarity) APG09338.0 (51.3% identity, 63.83%similarity) APG06242.0 (50.71% identity, 63.68% similarity) APG00911.0US_2016_0355842_A1-189 (50.0% identity, 63.37% similarity) APG00854.0US_2016_0355842_A1-179 (48.28% identity, 61.33% similarity)US_8829279_B2-11 (47.12% identity, 57.93% similarity) APG05372.0 (46.25%identity, 52.06% similarity) APG05896.0 (41.67% identity, 53.68%similarity) APG09338.0 149 150 MTX 55, 60, 65, 70, 75, 80, 70, 75, 80,85, 90, APG06242.0 (62.9% identity, 74.45% similarity) 85, 90, 91, 92,93, 94, 91, 92, 93, 94, 95, APG00051.0 US_2016_0304898_A1-81 (53.68% 95,96, 97, 98, 99, 100 96, 97, 98, 99, 100 identity, 68.41% similarity)US_8829279_B2-11 (52.76% identity, 65.33% similarity) APG00293.0US_2016_0355842_A1-51 (52.35% identity, 66.17% similarity) APG09211.0(51.3% identity, 63.83% similarity) APG05896.0 (49.87% identity, 60.45%similarity) APG00854.0 US_2016_0355842_A1-179 (49.14% identity, 61.73%similarity) APG00911.0 US_2016_0355842_A1-189 (48.4% identity, 62.96%similarity) APG00170.0 US_2016_0355842_A1-13 (48.11% identity, 61.08%similarity) APG05372.0 (44.67% identity, 53.55% similarity) APG09441.0151 152 Cry 35, 40, 45, 50, 55, 60, 50, 55, 60, 65, 70, WP_048536362.1(34.77% identity, 47.25% 65, 70, 75, 80, 85, 90, 75, 80, 85, 90, 91,similarity) 91, 92, 93, 94, 95, 96, 92, 93, 94, 95, 96, APG00372.0US_2016_0311864_A1-150 97, 98, 99, 100 97, 98, 99, 100 (34.14% identity,45.47% similarity) APG00615.0 US_2016_0366881_A1-119 (33.71% identity,45.48% similarity) APG00723.0 US_2016_0311864_A1-209 (33.63% identity,46.7% similarity) APG08607.0 (33.1% identity, 47.09% similarity)APG09630.0 (33.04% identity, 45.45% similarity) APG00285.0US_2016_0311864_A1-118 (31.85% identity, 44.94% similarity) APG00363.0US_2016_0366881_A1-34 (31.31% identity, 45.2% similarity)US_8318900_B2-91 (30.94% identity, 47.07% similarity) APG00787.0 (30.3%identity, 42.24% similarity) APG00329.0 US_2016_0355842_A1-62 (30.26%identity, 44.93% similarity) APG08973.0 (30.17% identity, 44.56%similarity) APG09459.0 153 154, 155 MTX 75, 80, 85, 90, 91, 92, 85, 90,91, 92, 93, CA_2844913-118 (74.62% identity, 84.42% 93, 94, 95, 96, 97,98, 94, 95, 96, 97, 98, similarity) 99, 100 99, 100 WP_016123520.1(74.12% identity, 85.18% similarity) CA_2844913-122 (27.93% identity,41.31% similarity) APG09460.0 156 157 MTX 97, 98, 99, 100 98, 99, 100APG00416.0 US_2016_0355842_A1-82 (96.66% identity, 97.57% similarity)APG07606.0 (86.96% identity, 88.04% similarity) WP_072335847.1 (51.5%identity, 67.37% similarity) APG00793.0 US_2016_0355842_A1-170 (37.5%identity, 56.53% similarity) SDZ23421.1 (35.45% identity, 51.87%similarity) US_8829279_B2-11 (33.33% identity, 53.01% similarity)US_8461415_B2-36 (32.98% identity, 47.23% similarity) APG00451.0US_2016_0355842_A1-94 (32.48% identity, 51.57% similarity) APG00911.0US_2016_0355842_A1-189 (31.52% identity, 48.91% similarity) APG05615.0(30.79% identity, 48.59% similarity) APG06242.0 (30.73% identity, 46.61%similarity) WP_000844425.1 (30.51% identity, 48.31% similarity)APG09492.0 158 159 MTX 90, 91, 92, 93, 94, 95, 92, 93, 94, 95, 96,APG09055.0 (86.94% identity, 91.99% similarity) 96, 97, 98, 99, 100 97,98, 99, 100 APG00569.0 US_2016_0355842_A1-129 (77.45% identity, 86.35%similarity) APG00938.0 US_2016_0355842_A1-190 (76.79% identity, 86.9%similarity) APG04477.0 (76.04% identity, 85.8% similarity) APG00794.0US_2016_0355842_A1-171 (74.78% identity, 86.05% similarity) APG00563.0US_2016_0355842_A1-127 (70.92% identity, 82.2% similarity) APG00387.0US_2016_0366881_A1-45 (64.41% identity, 76.76% similarity) APG00590.0US_2016_0311864_A1-193 (59.88% identity, 73.16% similarity)WP_000794514.1 (59.35% identity, 71.81% similarity) APG00146.0US_2016_0304898_A1-166 (59.05% identity, 71.51% similarity)CA_2844913-102 (57.65% identity, 72.06% similarity) ANN35739.1 (57.65%identity, 71.76% similarity) ANN35530.1 (56.6% identity, 71.55%similarity) US_8318900_B2-78 (56.38% identity, 68.25% similarity)APG06228.0 (56.23% identity, 69.57% similarity) APG09155.0 (55.88%identity, 71.18% similarity) APG09604.0 160 161 Cry 75, 80, 85, 90, 91,92, 85, 90, 91, 92, 93, US_8318900_B2-88 (73.52% identity, 81.85% 93,94, 95, 96, 97, 98, 94, 95, 96, 97, 98, similarity) 99, 100 99, 100ADK66923.1 (60.56% identity, 69.76% similarity) US_8318900_B2-82 (60.1%identity, 69.21% similarity) APG03519.0 (59.02% identity, 67.32%similarity) APG03519.0 (59.02% identity, 67.32% similarity) APG05370.0(58.83% identity, 69.44% similarity) APG00310.0 US_2016_0366881_A1-4(58.42% identity, 68.15% similarity) APG00974.0 (57.59% identity, 66.62%similarity) APG00068.0 US_2016_0177333_A1-35 (57.21% identity, 66.74%similarity) US_2016_0017363_A1-32 (57.11% identity, 67.29% similarity)APG00054.0 US_2016_0304898_A1-84 (57.04% identity, 67.22% similarity)US_8461421_B2-98 (56.95% identity, 66.15% similarity) US_8461421_B2-91(56.78% identity, 67.01% similarity) US_2016_0017363_A1-33 (56.51%identity, 66.62% similarity) APG05930.0 (55.98% identity, 65.52%similarity) US_8759619_B2-19 (55.93% identity, 66.64% similarity)APG05311.0 (55.85% identity, 63.22% similarity) APG05311.0 (55.85%identity, 63.22% similarity) US_8318900_B2-81 (55.67% identity, 65.55%similarity) APG00673.0 (55.25% identity, 64.33% similarity)US_8759619_B2-27 (55.11% identity, 64.54% similarity) APG09768.0 162 163Bin 45, 50, 55, 60, 65, 70, 60, 65, 70, 75, 80, APG00221.0US_2016_0311864_A1-81 (44.31% 75, 80, 85, 90, 91, 92, 85, 90, 91, 92,93, identity, 56.16% similarity) 93, 94, 95, 96, 97, 98, 94, 95, 96, 97,98, APG00716.0 US_2016_0311864_A1-393 99, 100 99, 100 (43.75% identity,55.32% similarity) WP_033679178.1 (43.54% identity, 59.57% similarity)APG00591.0 US_2016_0311864_A1-194 (43.52% identity, 58.19% similarity)APG00376.0 US_2016_0311864_A1-153 (43.52% identity, 55.32% similarity)APG00065.0 US_2016_0177333_A1-31 (43.13% identity, 57.58% similarity)APG00412.0 US_2016_0311864_A1-173 (42.76% identity, 56.29% similarity)APG00203.0 US_2016_0311864_A1-69 (42.55% identity, 55.08% similarity)CA_2844913-147 (42.49% identity, 55.43% similarity) APG00419.0US_2016_0311864_A1-176 (42.35% identity, 55.06% similarity) APG09977.0(42.31% identity, 56.73% similarity) APG07334.0 (42.15% identity, 55.74%similarity) APG00353.0 US_2016_0311864_A1-144 (42.08% identity, 55.32%similarity) APG00301.0 US_2016_0311864_A1-131 (42.08% identity, 54.85%similarity) APG01363.0 (41.96% identity, 55.01% similarity) APG00300.0US_2016_0355842_A1-53 (41.88% identity, 56.94% similarity) APG00243.0US_2016_0311864_A1-94 (41.59% identity, 56.49% similarity) APG00806.0(41.4% identity, 56.05% similarity) WP_070144216.1 (41.26% identity,55.24% similarity) APG07491.0 (41.25% identity, 55.88% similarity)APG00237.0 US_2016_0311864_A1-90 (41.23% identity, 54.21% similarity)APG00459.0 US_2016_0311864_A1-181 (41.15% identity, 55.17% similarity)APG00308.0 US_2016_0311864_A1-135 (41.04% identity, 55.9% similarity)APG00385.0 US_2016_0311864_A1-157 (41.03% identity, 55.71% similarity)APG00249.0 US_2016_0311864_A1-96 (41.01% identity, 56.22% similarity)APG02572.0 (41.01% identity, 54.15% similarity) APG00407.0US_2016_0311864_A1-170 (40.88% identity, 56.58% similarity) APG00132.0US_2016_0311864_A1-31 (40.88% identity, 56.12% similarity) APG00724.0(40.77% identity, 55.35% similarity) APG00336.0 US_2016_0366881_A1-19(40.68% identity, 55.23% similarity) US_8318900_B2-72 (40.65% identity,53.12% similarity) APG05715.0 (40.56% identity, 55.71% similarity)APG02445.0 (40.56% identity, 55.24% similarity) WP_074651503.1 (40.47%identity, 57.41% similarity) APG00229.0 US_2016_0311864_A1-84 (40.41%identity, 52.74% similarity) APG00550.0 US_2016_0311864_A1-190 (40.4%identity, 55.36% similarity) APG00295.0 US_2016_0311864_A1-128 (40.18%identity, 51.34% similarity) APG04176.0 (40.0% identity, 54.42%similarity) APG09869.0 164 165, 166 MTX 85, 90, 91, 92, 93, 94, 90, 91,92, 93, 94, WP_016099228.1 (85.93% identity, 89.6% 95, 96, 97, 98, 99,95, 96, 97, 98, 99, similarity) 100 100 APG00693.0 (80.79% identity,89.02% similarity) US_8461415_B2-31 (80.12% identity, 87.46% similarity)APG00495.0 (75.0% identity, 82.74% similarity) EEM56710.1 (50.91%identity, 65.45% similarity) WP_008180054.1 (50.0% identity, 65.45%similarity) APG00049.0 US_2016_0304898_A1-78 (49.39% identity, 63.33%similarity) WP_000730625.1 (49.09% identity, 63.03% similarity)APG03746.0 (48.48% identity, 63.03% similarity) WP_000790613.1 (44.64%identity, 60.12% similarity) WP_044797732.1 (44.57% identity, 61.88%similarity) WP_050822474.1 (44.12% identity, 61.76% similarity)WP_000163136.1 (44.12% identity, 61.18% similarity) APG00013.0US_2016_0304898_A1-22 (43.82% identity, 61.76% similarity)WP_065212007.1 (43.82% identity, 61.47% similarity) WP_001036192.1(43.27% identity, 60.82% similarity) APG00484.0 US_2016_0355842_A1-105(42.73% identity, 58.72% similarity) APG00382.0 US_2016_0355842_A1-74(42.15% identity, 58.72% similarity) APG00234.0 US_2016_0304898_A1-207(42.06% identity, 60.29% similarity) US_8829279_B2-38 (41.69% identity,58.91% similarity) APG09953.0 167 168 MTX 90, 91, 92, 93, 94, 95, 93,94, 95, 96, 97, APG00359.0 US_2016_0355842_A1-68 (88.67% 96, 97, 98, 99,100 98, 99, 100 identity, 92.88% similarity) US_2016_0017363_A1-59(35.28% identity, 49.44% similarity) ANS51604.1 (35.0% identity, 49.17%similarity) US_2016_0017363_A1-63 (34.21% identity, 48.25% similarity)US_2016_0017363_A1-60 (34.17% identity, 48.06% similarity)US_2016_0017363_A1-62 (34.13% identity, 49.1% similarity) APG05653.0(33.81% identity, 51.99% similarity) APG02674.0 (33.8% identity, 47.65%similarity) US_2016_0017363_A1-61 (33.61% identity, 47.22% similarity)APG01420.0 (33.61% identity, 45.9% similarity) WP_001267112.1 (33.24%identity, 47.04% similarity) WP_043924590.1 (33.24% identity, 46.65%similarity) WP_061688274.1 (33.06% identity, 47.11% similarity)APG09726.0 (32.98% identity, 42.74% similarity) US_8461415_B2-32 (32.87%identity, 46.41% similarity) WP_075716686.1 (32.14% identity, 44.23%similarity) APG06364.0 (31.5% identity, 44.51% similarity) AGC39300.1(31.08% identity, 43.61% similarity) WP_075718641.1 (30.75% identity,45.43% similarity) WP_050595402.1 (30.4% identity, 43.47% similarity)WP_023524027.1 (30.16% identity, 42.93% similarity) APG09977.0 169 170Bin 94, 95, 96, 97, 98, 99, 96, 97, 98, 99, 100 APG00243.0US_2016_0311864_A1-94 (93.21% 100 identity, 95.82% similarity)APG00301.0 US_2016_0311864_A1-131 (89.06% identity, 94.79% similarity)APG00353.0 US_2016_0311864_A1-144 (85.42% identity, 91.93% similarity)APG00844.0 US_2016_0311864_A1-221 (84.86% identity, 92.43% similarity)APG00213.0 US_2016_0311864_A1-76 (84.5% identity, 91.47% similarity)APG00203.0 US_2016_0311864_A1-69 (84.11% identity, 90.89% similarity)APG00065.0 US_2016_0177333_A1-31 (83.66% identity, 88.61% similarity)APG00412.0 US_2016_0311864_A1-173 (81.12% identity, 88.52% similarity)APG00419.0 US_2016_0311864_A1-176 (80.9% identity, 87.19% similarity)APG00300.0 US_2016_0355842_A1-53 (80.4% identity, 87.44% similarity)APG07334.0 (80.25% identity, 86.58% similarity) WP_000839920.1 (80.2%identity, 87.06% similarity) APG00765.0 171 172, 173 MTX 85, 90, 91, 92,93, 94, 90, 91, 92, 93, 94, APG06727.0 (93.57% identity, 94.15%similarity) 95, 96, 97, 98, 99, 95, 96, 97, 98, 99, 100 100 APG09869.0(91.16% identity, 94.82% similarity) WP_016099228.1 (87.8% identity,89.02% similarity) APG00693.0 (80.55% identity, 87.84% similarity)US_8461415_B2-31 (80.49% identity, 86.89% similarity) OUB62697.1 (77.74%identity, 82.93% similarity) APG00495.0 (74.48% identity, 83.38%similarity) OUB77698.1 (70.33% identity, 79.82% similarity)WP_008180054.1 (49.85% identity, 64.35% similarity) APG03297.0 (48.8%identity, 62.65% similarity) WP_003290257.1 (48.8% identity, 62.65%similarity) APG01166.0 174 175, 176 Bin 75, 80, 85, 90, 91, 92, 80, 85,90, 91, 92, APG02572.0 (95.35% identity, 97.07% similarity) 93, 94, 95,96, 97, 98, 93, 94, 95, 96, 97, 99, 100 98, 99, 100 APG00988.0 (72.86%identity, 79.46% similarity) SDZ44649.1 (70.24% identity, 78.29%similarity) APG00261.0 US_2016_0311864_A1-100 (70.17% identity, 79.71%similarity) APG07491.0 (69.17% identity, 77.18% similarity) APG07247.0(68.46% identity, 79.22% similarity) APG00648.0 (68.46% identity, 78.48%similarity) WP_001258161.1 (67.48% identity, 79.46% similarity)CA_2844913-10 (67.24% identity, 79.22% similarity) WP_001258160.1(67.24% identity, 79.22% similarity) APG01488.0 177 MTX 93, 94, 95, 96,97, 98, 97, 98, 99, 100 APG00686.0 US_2016_0355842_A1-159 99, 100(92.97% identity, 96.64% similarity) APG04345.0 (92.19% identity, 95.8%similarity) APG00860.0 US_2016_0355842_A1-180 (29.11% identity, 46.11%similarity) APG00718.0 (27.92% identity, 45.87% similarity) APG04586.0(26.3% identity, 46.53% similarity) APG00586.0 US_2016_0355842_A1-135(26.21% identity, 45.87% similarity) US_8318900_B2-77 (25.78% identity,43.91% similarity) APG03101.0 (25.64% identity, 43.59% similarity)APG00420.0 US_2016_0355842_A1-85 (25.57% identity, 44.32% similarity)APG00323.0 US_2016_0355842_A1-60 (25.36% identity, 45.01% similarity)APG03067.0 178 179 MTX 94, 95, 96, 97, 98, 96, 97, 98, 99, 100APG03889.0 (95.21% identity, 97.18% similarity) 99, 100 APG05791.0(94.65% identity, 95.77% similarity) APG00429.0 US_2016_0366881_A1-53(93.52% identity, 95.21% similarity) APG00994.0 US_2016_0355842_A1-198(58.95% identity, 73.0% similarity) AQM56946.1 (58.4% identity, 72.73%similarity) APG02557.0 (57.42% identity, 73.08% similarity) APG04947.0180 181 MTX 99, 100 100 APG08509.0 (98.73% identity, 100.0% similarity)WP_077572519.1 (92.09% identity, 93.04% similarity) WP_023465358.1(74.05% identity, 82.59% similarity) WP_039843228.1 (70.89% identity,80.7% similarity) WP_058177796.1 (70.89% identity, 80.38% similarity)WP_015967180.1 (70.57% identity, 80.7% similarity) WP_015730144.1(57.86% identity, 73.27% similarity) EP_1770171-493 (48.73% identity,55.06% similarity) APG03604.0 (33.75% identity, 48.92% similarity)SDI38744.1 (33.44% identity, 48.92% similarity) APG00429.0US_2016_0366881_A1-53 (98.28% APG05791.0 182 183 MTX 99, 100 100identity, 99.14% similarity) APG03067.0 (94.65% identity, 95.77%similarity) APG03889.0 (92.96% identity, 95.21% similarity) APG00994.0US_2016_0355842_A1-198 (60.22% identity, 75.35% similarity) AQM56946.1(59.38% identity, 75.07% similarity) APG02557.0 (56.87% identity, 72.53%similarity) APG05956.0 184 185 MTX 95, 96, 97, 98, 97, 98, 99, 100APG09460.0 (96.35% identity, 98.48% similarity) 99, 100 APG00416.0US_2016_0355842_A1-82 (94.53% identity, 96.35% similarity) APG07606.0(84.78% identity, 86.68% similarity) WP_072335847.1 (49.1% identity,66.47% similarity) APG00793.0 US_2016_0355842_A1-170 (36.36% identity,55.4% similarity) SDZ23421.1 (33.71% identity, 50.0% similarity)US_8461415_B2-36 (32.89% identity, 46.84% similarity) APG00451.0US_2016_0355842_A1-94 (32.51% identity, 49.73% similarity)US_8829279_B2-11 (32.24% identity, 51.37% similarity) APG06242.0 (30.21%identity, 45.05% similarity) APG06419.0 186 187 Cry 75, 80, 85, 90, 91,92, 85, 90, 91, 92, 93, APG09604.0 (95.38% identity, 96.69% similarity)93, 94, 95, 96, 97, 98, 94, 95, 96, 97, 98, 99, 100 99, 100US_8318900_B2-88 (73.02% identity, 81.91% similarity) ADK66923.1 (60.65%identity, 69.79% similarity) US_8318900_B2-82 (60.56% identity, 69.68%similarity) APG05370.0 (58.36% identity, 69.05% similarity) APG00310.0US_2016_0366881_A1-4 (57.89% identity, 66.86% similarity) APG03519.0(57.78% identity, 66.14% similarity) APG03519.0 (57.78% identity, 66.14%similarity) US_2016_0017363_A1-32 (57.44% identity, 67.17% similarity)APG00054.0 US_2016_0304898_A1-84 (57.32% identity, 67.12% similarity)APG06727.0 188 189, 190 MTX 80, 85, 90, 91, 92, 93, 90, 91, 92, 93, 94,APG00765.0 (93.57% identity, 94.15% similarity) 94, 95, 96, 97, 98, 99,95, 96, 97, 98, 99, 100 100 APG09869.0 (88.01% identity, 91.81%similarity) WP_016099228.1 (84.5% identity, 85.67% similarity)APG00693.0 (78.13% identity, 85.13% similarity) US_8461415_B2-31 (78.07%identity, 84.5% similarity) OUB62697.1 (74.85% identity, 79.82%similarity) APG00495.0 (71.79% identity, 80.34% similarity) OUB77698.1(67.81% identity, 76.92% similarity) WP_003290257.1 (48.12% identity,62.03% similarity) WP_008180054.1 (48.12% identity, 62.03% similarity)APG03297.0 (48.12% identity, 61.16% similarity) APG09329.0 191 192, 193MTX 35, 40, 45, 50, 55, 60, 50, 55, 60, 65, 70, APG08330.0 (98.11%identity, 98.65% similarity) 65, 70, 75, 80, 85, 90, 75, 80, 85, 90, 91,91, 92, 93, 94, 95, 96, 92, 93, 94, 95, 96, 97, 98, 99, 100 97, 98, 99,100 APG07235.0 (41.54% identity, 57.21% similarity) US_8461415_B2-32(33.51% identity, 48.26% similarity) WP_001267112.1 (33.51% identity,47.72% similarity) WP_078401252.1 (32.9% identity, 50.13% similarity)US_2016_0017363_A1-59 (32.64% identity, 49.61% similarity) ANS51604.1(32.64% identity, 49.35% similarity) WP_075718641.1 (32.55% identity,48.18% similarity) AGC39300.1 (32.42% identity, 48.88% similarity)US_2016_0017363_A1-60 (32.11% identity, 49.09% similarity) APG03898.0194 195, 196 MTX 85, 90, 91, 92, 93, 94, 90, 91, 92, 93, 94, APG09869.0(90.83% identity, 95.11% similarity) 95, 96, 97, 98, 95, 96, 97, 98, 99,100 99, 100 APG00765.0 (90.55% identity, 93.6% similarity) APG06727.0(87.72% identity, 90.64% similarity) WP_016099228.1 (87.54% identity,91.28% similarity) US_8461415_B2-31 (82.41% identity, 88.89% similarity)APG00693.0 US_2017_0175134_A1-106 (81.85% identity, 89.54% similarity)WP_088114034.1 (80.37% identity, 85.36% similarity) APG00495.0US_2017_0175134_A1-12 (75.76% identity, 84.55% similarity)WP_086390045.1 (71.52% identity, 80.91% similarity) WP_008180054.1(50.93% identity, 66.98% similarity) APG06237.0 197 Bin 93, 94, 95, 96,97, 98, 96, 97, 98, 99, 100 APG08195.0 (94.85% identity, 97.3%similarity) 99, 100 APG07868.0 (92.63% identity, 96.31% similarity)APG00178.0 US_2016_0311864_A1-54 (92.63% identity, 95.82% similarity)APG00727.0 US_2016_0311864_A1-210 (90.66% identity, 93.61% similarity)APG00315.0 US_2016_0311864_A1-137 (89.46% identity, 93.63% similarity)APG00449.0 US_2016_0366881_A1-71 (29.21% identity, 45.33% similarity)APG00729.0 US_2017_0175134_A1-142 (29.01% identity, 42.2% similarity)APG00532.0 US_2016_0311864_A1-189 (28.47% identity, 44.71% similarity)APG00380.0 US_2016_0366881_A1-43 (28.3% identity, 48.11% similarity)APG00649.0 US_2017_0175134_A1-75 (27.95% identity, 45.91% similarity)APG04021.0 198 199, 200 MTX 50, 55, 60, 65, 70, 75, 65, 70, 75, 80, 85,APG09100.0 (97.13% identity, 98.85% similarity) 80, 85, 90, 91, 92, 93,90, 91, 92, 93, 94, 94, 95, 96, 97, 98, 99, 95, 96, 97, 98, 99, 100 100APG04586.0 (49.01% identity, 63.38% similarity) US_8318900_B2-77 (48.47%identity, 62.95% similarity) APG04627.0 201 Bin 95, 96, 97, 98, 99, 10097, 98, 99, 100 APG00178.0 US_2016_0311864_A1-54 (94.1% identity, 96.56%similarity) APG06237.0 (93.37% identity, 96.31% similarity) APG07868.0(93.37% identity, 95.82% similarity) APG08195.0 (93.37% identity, 95.82%similarity) APG00727.0 US_2016_0311864_A1-210 (90.42% identity, 93.86%similarity) APG00315.0 US_2016_0311864_A1-137 (88.45% identity, 92.63%similarity) APG05411.0 202 203 Bin 91, 92, 93, 94, 95, 96, 94, 95, 96,97, 98, APG01363.0 (94.94% identity, 95.95% similarity) 97, 98, 99, 10099, 100 WP_098205870.1 (92.41% identity, 95.7% similarity) APG07334.0(90.89% identity, 94.43% similarity) APG00229.0 US_2016_0311864_A1-84(90.35% identity, 93.56% similarity) APG00419.0 US_2016_0311864_A1-176(90.2% identity, 93.22% similarity) WP_098634587.1 (90.2% identity,93.22% similarity) APG00844.0 US_2016_0311864_A1-221 (86.58% identity,90.13% similarity) APG00301.0 US_2016_0311864_A1-131 (85.82% identity,89.37% similarity) APG00203.0 US_2016_0311864_A1-69 (85.06% identity,88.86% similarity) WP_098475832.1 (85.06% identity, 88.86% similarity)APG00353.0 US_2016_0311864_A1-144 (85.06% identity, 88.61% similarity)WP_099003921.1 (85.06% identity, 88.61% similarity) APG08768.0 204 205MTX 85, 90, 91, 92, 93, 94, 90, 91, 92, 93, 94, APG03324.0 (91.37%identity, 94.94% similarity) 95, 96, 97, 98, 99, 100 95, 96, 97, 98, 99,100 APG00586.0 US_2016_0355842_A1-135 (83.48% identity, 88.5%similarity) APG00420.0 US_2016_0355842_A1-85 (82.94% identity, 89.12%similarity) APG00323.0 US_2016_0355842_A1-60 (82.2% identity, 88.13%similarity) APG03101.0 (71.81% identity, 81.31% similarity)

i. Classes of Pesticidal Proteins

The pesticidal proteins provided herein and the nucleotide sequencesencoding them are useful in methods for impacting pests. That is, thecompositions and methods of the invention find use in agriculture forcontrolling or killing pests, including pests of many crop plants. Thepesticidal proteins provided herein are toxin proteins from bacteria andexhibit activity against certain pests. The pesticidal proteins are fromseveral classes of toxins including Cry, Cyt, BIN, Mtx toxins. See, forexample, Table 1 for the specific protein classifications of the variousSEQ ID NOS provided herein. In addition, reference is made throughoutthis disclosure to Pfam database entries. The Pfam database is adatabase of protein families, each represented by multiple sequencealignments and a profile hidden Markov model. Finn et al. (2014) Nucl.Acid Res. Database Issue 42:D222-D230.

Bacillus thuringiensis (Bt) is a gram-positive bacterium that producesinsecticidal proteins as crystal inclusions during its sporulation phaseof growth. The proteinaceous inclusions of Bacillus thuringiensis (Bt)are called crystal proteins or δ-endotoxins (or Cry proteins), which aretoxic to members of the class Insecta and other invertebrates.Similarly, Cyt proteins are parasporal inclusion proteins from Bt thatexhibits hemolytic (Cytolitic) activity or has obvious sequencesimilarity to a known Cyt protein. These toxins are highly specific totheir target organism, are innocuous to humans, vertebrates, and plants.

The structure of the Cry toxins reveals five conserved amino acidblocks, concentrated mainly in the center of the domain or at thejunction between the domains. The Cry toxin consists of three domains,each with a specific function. Domain I is a seven α-helix bundle inwhich a central helix is completely surrounded by six outer helices.This domain is implicated in channel formation in the membrane. DomainII appears as a triangular column of three anti-parallel β-sheets, whichare similar to antigen-binding regions of immunoglobulins. Domain IIIcontains anti-parallel β-strands in a β sandwich form. The N-terminalpart of the toxin protein is responsible for its toxicity andspecificity and contains five conserved regions. The C-terminal part isusually highly conserved and probably responsible for crystal formation.See, for example, U.S. Pat. No. 8,878,007.

Strains of B. thuringiensis show a wide range of specificity againstdifferent insect orders (Lepidoptera, Diptera, Coleoptera, Hymenoptera,Homoptera, Phthiraptera or Mallophaga, and Acari) and otherinvertebrates (Nemathelminthes, Platyhelminthes, and Sarocomastebrates).The cry proteins have been classified into groups based on toxicity tovarious insect and invertebrate groups. Generally, Cry I demonstratestoxicity to lepidopterans, Cry II to lepidopterans and dipterans, CryIIIto coleopterans, Cry IV to dipterans, and Cry V and Cry VI to nematodes.New Cry proteins can be identified and assigned to a Cry group based onamino acid identity. See, for example, Bravo, A. (1997) J. of Bacteriol.179:2793-2801; Bravo et al. (2013) Microb. Biotechnol. 6:17-26, hereinincorporated by reference.

Over 750 different cry gene sequences have been classified into 73groups (Cry1-Cry73), with new members of this gene family continuing tobe discovered (Crickmore et al. (2014) www.btnomenclature.info/). Thecry gene family consists of several phylogentically non-related proteinfamilies that may have different modes of action: the family ofthree-domain Cry toxins, the family of mosquitocidal Cry toxins, thefamily of the binary-like toxins, and the Cyt family of toxins (Bravo etal., 2005). Some Bt strains produce additional insecticidal toxins, theVIP toxins. See, also, Cohen et al. (2011) J. Mol. Biol. 413:4-814;Crickmore et al. (2014) Bacillus thuringiensis toxin nomenclature, foundon the world wide web atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/;Crickmore et al. (1988) Microbiol. Mol. Biol. Rev. 62: 807-813; Gill etal. (1992) Ann. Rev. Entomol. 37: 807-636; Goldbert et al. (1997) Appl.Environ. Microbiol. 63:2716-2712; Knowles et al. (1992) Proc. R. Soc.Ser. B. 248: 1-7; Koni et al. (1994) Microbiology 140: 1869-1880; Lailaket al. (2013) Biochem. Biophys. Res. Commun. 435: 216-221; Lopez-Diaz etal. (2013) Environ. Microbiol. 15: 3030-3039; Perez et al. (2007) Cell.Microbiol. 9: 2931-2937; Promdonkoy et al. (2003) Biochem. 1 374:255-259; Rigden (2009) FEBS Lett. 583: 1555-1560; Schnepf et al. (1998)Microbiol. Mol. Biol. Rev. 62: 775-806; Soberon et al. (2013) Peptides41: 87-93; Thiery et al. (1998) J. Am. Mosq. Control Assoc. 14: 472-476;Thomas et al. (1983) FEBS Lett. 154: 362-368; Wirth et al. (1997) Proc.Natl. Acad. Sci. U.S.A. 94: 10536-10540; Wirth et al (2005) Appl.Environ. Microbiol. 71: 185-189; and, Zhang et al. (2006) Biosci.Biotechnol. Biochem. 70: 2199-2204; each of which is herein incorporatedby reference in their entirety.

Cyt designates a parasporal crystal inclusion protein from Bacillusthuringiensis with cytolytic activity, or a protein with sequencesimilarity to a known Cyt protein. (Crickmore et al. (1998) Microbiol.Mol. Biol. Rev. 62: 807-813). The gene is denoted by cyt. These proteinsare different in structure and activity from Cry proteins (Gill et al.(1992) Annu. Rev. Entomol. 37: 615-636). The Cyt toxins were firstdiscovered in B. thuringiensis subspecies israelensis (Goldberg et al.(1977) Mosq. News. 37: 355-358). There are 3 Cyt toxin familiesincluding 11 holotype toxins in the current nomenclature (Crickmore etal. (2014) Bacillus thuringiensis toxin nomenclature found on the worldwide web atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/). The majorityof the B. thuringiensis isolates with cyt genes show activity againstdipteran insects (particularly mosquitoes and black flies), but thereare also cyt genes that have been described in B. thuringiensis strainstargeting lepidopteran or coleopteran insects (Guerchicoff et al. (1997)Appl. Environ. Microbiol. 63: 2716-2721).

The structure of Cyt2A, solved by X-ray crystallography, shows a singledomain where two outer layers of a-helix wrap around a mixed β-sheet.Further available crystal structures of Cyt toxins support a conservedα-β structural model with two α-helix hairpins flanking a β-sheet corecontaining seven to eight β-strands. (Cohen et al. (2011) J. Mol. Biol.413: 80 4-814) Mutagenic studies identified β-sheet residues as criticalfor toxicity, while mutations in the helical domains did not affecttoxicity (Adang et al.; Diversity of Bacillus thuringiensis CrystalToxins and Mechanism of Action. In: T. S. Dhadialla and S. S. Gill, eds,Advances in Insect Physiology, Vol. 47, Oxford: Academic Press, 2014,pp. 39-87.) The representative domain of the Cyt toxin is a δ-endotoxin,Bac_thur_toxin (Pfam PF01338).

There are multiple proposed models for the mode of action of Cyt toxins,and it is still an area of active investigation. Some Cyt proteins(Cyt1A) have been shown to require the presence of accessory proteinsfor crystallization. Cyt1A and Cyt2A protoxins are processed bydigestive proteases at the same sites in the N- and C-termini to astable toxin core. Cyt toxins then interact with non-saturated membranelipids, such as phosphatidylcholine, phosphatidylethanolamine, andsphingomyelin. For Cyt toxins, pore-formation and detergent-likemembrane disruption have been proposed as non-exclusive mechanisms; andit is generally accepted that both may occur depending on toxinconcentration, with lower concentrations favoring oligomeric pores andhigher concentrations leading to membrane breaks. (Butko (2003) Appl.Environ. Microbiol. 69: 2415-2422) In the pore-formation model, the Cyttoxin binds to the cell membrane, inducing the formation ofcation-selective channels in the membrane vesicles leading tocolloid-osmotic lysis of the cell. (Knowles et al. (1989) FEBS Lett.244: 259-262; Knowles et al. (1992) Proc. R. Soc. Ser. B. 248: 1-7 andPromdonkoy et al. (2003) Biochem. 1 374: 255-259). In the detergentmodel, there is a nonspecific aggregation of the toxin on the surface ofthe lipid bilayer leading to membrane disassembly and cell death. (Butko(2003) supra; Manceva et al. (2005) Biochem. 44: 589-597).

Multiple studies have shown synergistic activity between Cyt toxins andother B. thuringiensis toxins, particularly the Cry, Bin, and Mtxtoxins. This synergism has even been shown to overcome an insect'sresistance to the other toxin. (Wirth 1997, Wirth 2005, Thiery 1998,Zhang 2006) The Cyt synergistic effect for Cry toxins is proposed toinvolve Cyt1A binding to domain II of Cry toxins in solution or on themembrane plane to promote formation of a Cry toxin pre-pore oligomer.Formation of this oligomer is independent of the Cyt oligomerization,binding or insertion. (Lailak 2013, Perez 2007, Lopez-Diaz 2013)

A number of pesticidal proteins unrelated to the Cry proteins areproduced by some strains of B. thuringiensis and B. cereus duringvegetative growth (Estruch et al. (1996) Proc Natl Acad Sci USA93:5389-5394; Warren et al. (1994) WO 94/21795). These vegetativeinsecticidal proteins, or Vips, do not form parasporal crystal proteinsand are apparently secreted from the cell. The Vips are presentlyexcluded from the Cry protein nomenclature because they are notcrystal-forming proteins. The term VIP is a misnomer in the sense thatsome B. thuringiensis Cry proteins are also produced during vegetativegrowth as well as during the stationary and sporulation phases, mostnotably Cry3Aa. The location of the Vip genes in the B. thuringiensisgenome has been reported to reside on large plasmids that also encodecry genes (Mesrati et al. (2005) FEMS Microbiol. Lett. 244(2):353-8). Aweb-site for the nomenclature of Bt toxins can be found on the worldwide web at lifesci.sussex.ac.uk with the path“/home/Neil_Crickmore/Bt/” and at: “btnomenclature.info/”. See also,Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62(3):775-806. Suchreferences are herein incorporated by reference.

To date four categories of Vips have been identified. Some Vip genesform binary two-component protein complexes; an “A” component is usuallythe “active” portion, and a “B” component is usually the “binding”portion. (Pfam pfam.xfam.org/family/PF03495). The Vip1 and Vip4 proteinsgenerally contain binary toxin B protein domains. Vip2 proteinsgenerally contain binary toxin A protein domains.

The Vip1 and Vip2 proteins are the two components of a binary toxin thatexhibits toxicity to coleopterans. Vip1Aa1 and Vip2Aa1 are very activeagainst corn rootworms, particularly Diabrotica virgifera and Diabroticalongicornis (Han et al. (1999) Nat. Struct. Biol. 6:932-936; Warren G W(1997) “Vegetative insecticidal proteins: novel proteins for control ofcorn pests” In: Carozzi N B, Koziel M (eds) Advances in insect control,the role of transgenic plants; Taylor & Francis Ltd, London, pp 109-21).The membrane-binding 95 kDa Vip1 multimer provides a pathway for the 52kDa vip2 ADP-ribosylase to enter the cytoplasm of target western cornrootworm cells (Warren (1997) supra). The NAD-dependentADP-ribosyltransferase Vip2 likely modifies monomeric actin at Arg177 toblock polymerization, leading to loss of the actin cytoskeleton andeventual cell death due to the rapid subunit ex-change within actinfilaments in vivo (Carlier M. F. (1990) Adv. Biophys. 26:51-73).

Like Cry toxins, activated Vip3A toxins are pore-forming proteinscapable of making stable ion channels in the membrane (Lee et al. (2003)Appl. Environ. Microbiol. 69:4648-4657). Vip3 proteins are activeagainst several major lepidopteran pests (Rang et al. (2005) Appl.Environ. Microbiol. 71(10):6276-6281; Bhalla et al. (2005) FEMSMicrobiol. Lett. 243:467-472; Estruch et al. (1998) WO 9844137; Estruchet al. (1996) Proc Natl Acad Sci USA 93:5389-5394; Selvapandiyan et al.(2001) Appl. Environ Microbiol. 67:5855-5858; Yu et al. (1997) Appl.Environ Microbiol. 63:532-536). Vip3A is active against Agrotis ipsilon,Spodoptera frugiperda, Spodoptera exigua, Heliothis virescens, andHelicoverpa zea (Warren et al. (1996) WO 96/10083; Estruch et al. (1996)Proc Natl Acad Sci USA 93:5389-5394). Like Cry toxins, Vip3A proteinsmust be activated by proteases prior to recognition at the surface ofthe midgut epithelium of specific membrane proteins different from thoserecognized by Cry toxins.

The MTX family of toxin proteins is characterized by the presence of aconserved domain, ETX_MTX2 (pfam 03318). Members of this family sharesequence homology with the mosquitocidal toxins Mtx2 and Mtx3 fromBacillus sphaericus, as well as with the epsilon toxin ETX fromClostridium perfringens (Cole et al. (2004) Nat. Struct. Mol. Biol. 11:797-8; Thanabalu et al. (1996) Gene 170:85-9). The MTX-like proteins arestructurally distinct from the three-domain Cry toxins, as they have anelongated and predominately β-sheet-based structure. However, similar tothe three-domain toxins, the MTX-like proteins are thought to form poresin the membranes of target cells (Adang et al. (2014) supra). Unlike thethree-domain Cry proteins, the MTX-like proteins are much smaller inlength, ranging from 267 amino acids (Cry23) to 340 amino acids (Cry15A.

To date, only 15 proteins belonging to the family of MTX-like toxinshave been assigned Cry names, making this a relatively small classcompared to the three-domain Cry family (Crickmore et al. (2014) supra;Adang et al. (2014) supra). The members of the MTX-like toxin familyinclude Cry15, Cry23, Cry33, Cry38, Cry45, Cry46, Cry51, Cry60A, Cry60B,and Cry64. This family exhibits a range of insecticidal activity,including activity against insect pests of the Lepidopteran andColeopteran orders. Some members of this family may form binarypartnerships with other proteins, which may or may not be required forinsecticidal activity.

Cry15 is a 34 kDA protein that was identified in Bacillus thuringiensisserovar thompsoni HD542; it occurs naturally in a crystal together withan unrelated protein of approximately 40 kDa. The gene encoding Cry15and its partner protein are arranged together in an operon. Cry15 alonehas been shown to have activity against lepidopteran insect pestsincluding Manduca sexta, Cydia pomonella, and Pieris rapae, with thepresence of the 40 kDA protein having been shown to increase activity ofCry15 only against C. pomonella (Brown K. and Whiteley H. (1992) J.Bacteriol. 174:549-557; Naimov et al. (2008) Appl. Environ. Microbiol.74:7145-7151). Further studies are needed to elucidate the function ofthe partner protein of Cry15. Similarly, Cry23 is a 29 kDA protein thathas been shown to have activity against the coleopteran pests Triboliumcastaneum and Popillia japonica together with its partner protein Cry37(Donovan et al. (2000) U.S. Pat. No. 6,063,756).

New members of the MTX-like family are continuing to be identified. AnETX_MTX toxin gene was recently identified in the genome of Bacillusthuringiensis serovar tolworthi strain Na205-3. This strain was found tobe toxic against the lepidpoteran pest Helicoverpa armigera, and it alsocontained homologs of Cry1, Cry11, Vip1, Vip2, and Vip3 (Palma et al.(2014) Genome Announc. 2(2): e00187-14. Published online Mar. 13, 2014at doi: 10.1128/genomeA.00187-14; PMCID: PMC3953196). Because theMTX-like proteins have a unique domain structure relative to thethree-domain Cry proteins, they are believed to possess a unique mode ofaction, thereby making them a valuable tool in insect control and thefight against insect resistance.

Bacterial cells produce large numbers of toxins with diverse specificityagainst host and non-host organisms. Large families of binary toxinshave been identified in numerous bacterial families, including toxinsthat have activity against insect pests. (Poopathi and Abidha (2010) J.Physiol. Path. 1(3): 22-38). Lysinibacillus sphaericus (Ls), formerlyBacillus sphaericus, (Ahmed et al. (2007) Int. J. Syst. Evol. Microbiol.57:1117-1125) is well-known as an insect biocontrol strain. Ls producesseveral insecticidal proteins, including the highly potent binarycomplex BinA/BinB. This binary complex forms a parasporal crystal in Lscells and has strong and specific activity against dipteran insects,specifically mosquitos. In some areas, insect resistance to existing Lsmosquitocidal strains has been reported. The discovery of new binarytoxins with different target specificity or the ability to overcomeinsect resistance is of significant interest.

The Ls binary insecticidal protein complex contains two majorpolypeptides, a 42 kDa polypeptide and a 51 kDa polypepdide, designatedBinA and BinB, respectively (Ahmed et al. (2007) supra). The twopolypeptides act synergistically to confer toxicity to their targets.Mode of action involves binding of the proteins to receptors in thelarval midgut. In some cases, the proteins are modified by proteasedigestion in the larval gut to produce activated forms. The BinBcomponent is thought to be involved in binding, while the BinA componentconfers toxicity (Nielsen-LeRoux et al. (2001) Appl. Environ. Microbiol.67(11):5049-5054). When cloned and expressed separately, the BinAcomponent is toxic to mosquito larvae, while the BinB component is not.However, co-administration of the proteins markedly increases toxicity(Nielsen-LeRoux et al. (2001) supra).

A small number of Bin protein homologs have been described frombacterial sources. Priest et al. (1997) Appl. Environ. Microbiol.63(4):1195-1198 describe a hybridization effort to identify new Lsstrains, although most of the genes they identified encoded proteinsidentical to the known BinA/BinB proteins. The BinA protein contains adefined conserved domain known as the Toxin 10 superfamily domain. Thistoxin domain was originally defined by its presence in BinA and BinB.The two proteins both have the domain, although the sequence similaritybetween BinA and BinB is limited in this region (<40%). The Cry49Aaprotein, which also has insecticidal activity, also has this domain(described below).

The Cry48Aa/Cry49Aa binary toxin of Ls has the ability to kill Culexquinquefasciatus mosquito larvae. These proteins are in a proteinstructural class that has some similarity to the Cry protein complex ofBacillus thuringiensis (Bt), a well-known insecticidal protein family.The Cry34/Cry35 binary toxin of Bt is also known to kill insects,including Western corn rootworm, a significant pest of corn. Cry34, ofwhich several variants have been identified, is a small (14 kDa)polypeptide, while Cry35 (also encoded by several variants) is a 44 kDapolypeptide. These proteins have some sequence homology with theBinA/BinB protein group and are thought to be evolutionarily related(Ellis et al. (2002) Appl. Environ. Microbiol. 68(3):1137-1145).

Phosphoinositide phospholipase C proteins (PI-PLC; alsophosphotidylinositol phospholipase C) are members of the broader groupof phospholipase C proteins. Many of these proteins play important rolesin signal transduction as part of normal cell physiology. Severalimportant bacterial toxins also contain domains with similarity to theseproteins (Titball, R. W. (1993) Microbiological Reviews. 57(2):347-366).Importantly, these proteins are implicated in signal amplificationduring intoxication of insect cells by Bt Cry proteins (Valaitis, A. P.(2008) Insect Biochemistry and Molecular Biology. 38: 611-618).

The PI-PLC toxin class occurs in Bacillus isolates, commonly seen inco-occurrence with homologs to other described toxin classes, such asBinary Toxins. This class of sequences has homology tophosphatidylinositol phosphodiesterases (also referred to asphosphatidylinositol-specific phospholipase C-PI-PLC). The crystalstructure and its active site were solved for B. cereus PI-PLC by Heinzet al (Heinz, et. al., (1995) The EMBO Journal. 14(16): 3855-3863). Theroles of the B. cereus PI-PLC active site amino acid residues incatalysis and substrate binding were investigated by Gässier et al usingsite-directed mutagenesis, kinetics, and crystal structure analysis(Gässier, et. al., (1997) Biochemistry. 36(42):12802-13).

These PI-PLC toxin proteins contain a PLC-like phosphodiesterase, TIMbeta/alpha-barrel domain (IPR017946) and/or a Phospholipase C,phosphatidylinositol-specific, X domain (IPR000909) (also referred to asthe PI-PLC X-box domain). We have also seen proteins with these domainsin combination with other typical Bacillus protein toxin domains. Thislist includes most commonly a lectin domain (IPR000772), a sugar-bindingdomain that can be present in one or more copies and is thought to bindcell membranes, as well as the Insecticidal crystal toxin (IPR008872)(also referred to as Toxin10 or P42), which is the defining domain ofthe Binary Toxin. An example of this domain structure is in sequenceAPG00732.

Previously, toxins of this PI-PLC class were defined in U.S. Pat. No.8,318,900 B2 SEQ ID NOs 30 (DNA) and 79 (amino acid), in U.S. PatentPublication No. 20110263488A1 SEQ ID NOs 8 (DNA) and 9 (amino acid), andin U.S. Pat. No. 8,461,421B2 SEQ ID NOs 3 (DNA) and 63 (amino acid).

Provided herein are pesticidal proteins from these classes of toxins.The pesticidal proteins are classified by their structure, homology toknown toxins and/or their pesticidal specificity.

ii. Variants and Fragments of Pesticidal Proteins and PolynucleotidesEncoding the Same

Pesticidal proteins or polypeptides of the invention include those setforth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202,203, 204, and/or 205 and fragments and variants thereof. By “pesticidaltoxin” or “pesticidal protein” or “pesticidal polypeptide” is intended atoxin or protein or polypeptide that has activity against one or morepests, including, insects, fungi, nematodes, and the like such that thepest is killed or controlled.

An “isolated” or “purified” polypeptide or protein, or biologicallyactive portion thereof, is substantially or essentially free fromcomponents that normally accompany or interact with the polypeptide orprotein as found in its naturally occurring environment. Thus, anisolated or purified polypeptide or protein is substantially free ofother cellular is material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. A protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) ofcontaminating protein. When the protein of the invention or biologicallyactive portion thereof is recombinantly produced, optimally culturemedium represents less than about 30%, 20%, 10%, 5%, or 1% (by dryweight) of chemical precursors or non-protein-of-interest chemicals.

The term “fragment” refers to a portion of a polypeptide sequence of theinvention. “Fragments” or “biologically active portions” includepolypeptides comprising a sufficient number of contiguous amino acidresidues to retain the biological activity, i.e., have pesticidalactivity. Fragments of the pesticidal proteins include those that areshorter than the full-length sequences, either due to the use of analternate downstream start site, or due to processing that produces ashorter protein having pesticidal activity. Processing may occur in theorganism the protein is expressed in, or in the pest after ingestion ofthe protein. Examples of fragments of the proteins can be found inTable 1. A biologically active portion of a pesticidal protein can be apolypeptide that is, for example, 10, 25, 50, 100, 150, 200, 250 or moreamino acids in length of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, and/or 205. Such biologicallyactive portions can be prepared by recombinant techniques and evaluatedfor pesticidal activity. As used here, a fragment comprises at least 8contiguous amino acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205.

Bacterial genes, including those encoding the pesticidal proteinsdisclosed herein, quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present invention andmay be used in the methods disclosed herein. It will be understood that,when expressed in plants, it will be necessary to alter the alternatestart codon to ATG for proper translation.

In various embodiments the pesticidal proteins provided herein includeamino acid sequences deduced from the full-length nucleotide sequencesand amino acid sequences that are shorter than the full-length sequencesdue to the use of an alternate downstream start site. Thus, thenucleotide sequence of the invention and/or vectors, host cells, andplants comprising the nucleotide sequence of the invention (and methodsof making and using the nucleotide sequence of the invention) maycomprise a nucleotide sequence encoding an alternate start site.

It is recognized that modifications may be made to the pesticidalpolypeptides provided herein creating variant proteins. Changes designedby man may be introduced through the application of site-directedmutagenesis techniques. Alternatively, native, as yet-unknown or as yetunidentified polynucleotides and/or polypeptides structurally and/orfunctionally-related to the sequences disclosed herein may also beidentified that fall within the scope of the present invention.Conservative amino acid substitutions may be made in nonconservedregions that do not alter the function of the pesticidal proteins.Alternatively, modifications may be made that improve the activity ofthe toxin. Modification of Cry toxins by domain III swapping hasresulted in some cases in hybrid toxins with improved toxicities againstcertain insect species. Thus, domain III swapping could be an effectivestrategy to improve toxicity of Cry toxins or to create novel hybridtoxins with toxicity against pests that show no susceptibility to theparental Cry toxins. Site-directed mutagenesis of domain II loopsequences may result in new toxins with increased insecticidal activity.Domain II loop regions are key binding regions of initial Cry toxinsthat are suitable targets for the mutagenesis and selection of Crytoxins with improved insecticidal properties. Domain I of the Cry toxinmay be modified to introduce protease cleavage sites to improve activityagainst certain pests. Strategies for shuffling the three differentdomains among large numbers of cry genes and high through outputbioassay screening methods may provide novel Cry toxins with improved ornovel toxicities.

As indicated, fragments and variants of the polypeptides disclosedherein will retain pesticidal activity. Pesticidal activity comprisesthe ability of the composition to achieve an observable effectdiminishing the occurrence or an activity of the target pest, includingfor example, bringing about death of at least one pest, or a noticeablereduction in pest growth, feeding, or normal physiological development.Such decreases in numbers, pest growth, feeding or normal developmentcan comprise any statistically significant decrease, including, forexample a decrease of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater. The pesticidalactivity against one or more of the various pests provided herein,including, for example, pesticidal activity against Coleoptera, Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Nematodes, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., or any other pest described herein. It is recognizedthat the pesticidal activity may be different or improved relative tothe activity of the native protein, or it may be unchanged, so long aspesticidal activity is retained. Methods for measuring pesticidalactivity are provide elsewhere herein. See also, Czapla and Lang (1990)J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

By “variants” is intended polypeptides having an amino acid sequencethat is at least about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% identical to the amino acid sequence of any ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, and/or 205 and retain pesticidal activity. Note, Table 1 providesnon-limiting examples of variant polypeptides (and polynucleotideencoding the same) for each of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, and/or 205. A biologically activevariant of a pesticidal polypeptide of the invention may differ by asfew as about 1-15 amino acid residues, as few as about 1-10, such asabout 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or asfew as 1 amino acid residue. In specific embodiments, the polypeptidescan comprise an N-terminal or a C-terminal truncation, which cancomprise at least a deletion of 10, 15, 20, 25, 30, 35, 40, 45, 50 aminoacids or more from either the N or C terminal of the polypeptide.

Table 2 provides protein domains found in SEQ ID NOs: 1-205 based onPFAM data. Both the domain description and the positions within a givenSEQ ID NO are provided in Table 2. In specific embodiments, the activevariant comprising any one of SEQ ID NOs: 1-205 can comprise at least70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to any one of SEQ ID NOs: 1-205 and further comprises at leastone of the conserved domain set forth in Table 2. For example, in oneembodiment, the active variant will comprise at least 70%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQID NO:1, and further comprises the native amino acids at positions 156to 343 and/or 48-143.

TABLE 2 Summary of PFAM domains in each of SEQ ID NOs: 1-205 SeqModification APG ID ID Type Accession Description Start Stop APG00837.01 PF14200 Ricin-type beta-trefoil lectin 48 143 domain-like PF05431Insecticidal Crystal Toxin, P42 156 343 APG01013.0 2 PF03945 deltaendotoxin, N-terminal 69 318 domain PF00555 delta endotoxin 324 508PF03944 delta endotoxin 518 650 PF14200 Ricin-type beta-trefoil lectin700 802 domain-like APG01013.1 3 3′ Truncation PF03945 delta endotoxin,N-terminal 69 318 domain PF00555 delta endotoxin 324 508 PF03944 deltaendotoxin 518 650 APG01155.0 4 no PFAM domains APG01155.1 5 Signalpeptide no PFAM removed domains APG01189.0 6 PF03318 Clostridium epsilontoxin 81 284 ETX/Bacillus mosquitocidal toxin MTX2 APG01189.1 7 Signalpeptide PF03318 Clostridium epsilon toxin 59 262 removed ETX/Bacillusmosquitocidal toxin MTX2 APG01363.0 8 PF05431 Insecticidal CrystalToxin, P42 201 390 APG01363.1 9 Signal peptide PF05431 InsecticidalCrystal Toxin, P42 172 361 removed APG01502.0 10 PF01338 Bacillusthuringiensis toxin 19 234 APG01502.1 11 Alternate start PF01338Bacillus thuringiensis toxin 19 234 APG01844.0 12 PF03945 deltaendotoxin, N-terminal 65 284 domain PF00555 delta endotoxin 290 499PF03944 delta endotoxin 510 645 APG01844.1 13 Alternate start PF03945delta endotoxin, N-terminal 62 281 domain PF00555 delta endotoxin 287496 PF03944 delta endotoxin 507 642 APG01844 14 no PFAM Split-Cry C-domains terminus (APG09587.0) APG01927.0 15 PF07691 PA14 domain 16 140PF03495 Clostridial binary toxin 186 597 B/anthrax toxin PA APG01969.016 PF03318 Clostridium epsilon toxin 106 314 ETX/Bacillus mosquitocidaltoxin MTX2 APG01969.1 17 Signal peptide PF03318 Clostridium epsilontoxin 73 281 removed ETX/Bacillus mosquitocidal toxin MTX2 APG01971.0 18PF01338 Bacillus thuringiensis toxin 33 220 APG01971.1 19 Alternatestart PF01338 Bacillus thuringiensis toxin 15 202 APG02013.0 20 PF03318Clostridium epsilon toxin 39 249 ETX/Bacillus mosquitocidal toxin MTX2APG02013.1 21 Alternate start PF03318 Clostridium epsilon toxin 5 215ETX/Bacillus mosquitocidal toxin MTX2 APG02060.0 22 PF03318 Clostridiumepsilon toxin 109 313 ETX/Bacillus mosquitocidal toxin MTX2 APG02060.123 Signal peptide PF03318 Clostridium epsilon toxin 75 280 removedETX/Bacillus mosquitocidal toxin MTX2 APG02123.0 24 no PFAM domainsAPG02123.1 25 Signal peptide no PFAM removed domains APG02403.0 26PF03945 delta endotoxin, N-terminal 78 314 domain PF03944 deltaendotoxin 528 666 APG02403.1 27 3′ Truncation PF03945 delta endotoxin,N-terminal 77 314 domain PF03944 delta endotoxin 528 666 APG02403 28PF14200 Ricin-type beta-trefoil lectin 360 459 Split-Cry C- domain-liketerminus (APG07980.0) APG02438.0 29 PF03945 delta endotoxin, N-terminal69 317 domain PF00555 delta endotoxin 326 401 PF03944 delta endotoxin526 657 APG02438.1 30 Alternate start PF03945 delta endotoxin,N-terminal 45 293 domain PF00555 delta endotoxin 302 377 PF03944 deltaendotoxin 502 633 APG02572.0 31 PF05431 Insecticidal Crystal Toxin, P42206 400 APG02572.1 32 Signal peptide PF05431 Insecticidal Crystal Toxin,P42 172 366 removed APG02630.0 33 PF03945 delta endotoxin, N-terminal236 367 domain PF03944 delta endotoxin 571 705 APG02630.1 34 Alternatestart PF03945 delta endotoxin, N-terminal 209 340 domain PF03944 deltaendotoxin 544 678 APG02630.2 35 Alternate start PF03945 delta endotoxin,N-terminal 209 340 domain PF03944 delta endotoxin 544 677 APG02647.0 36PF01338 Bacillus thuringiensis toxin 37 254 APG02647.1 37 Alternatestart PF01338 Bacillus thuringiensis toxin 37 254 APG02701.0 38 PF03318Clostridium epsilon toxin 93 316 ETX/Bacillus mosquitocidal toxin MTX2APG02701.1 39 Signal peptide PF03318 Clostridium epsilon toxin 59 281removed ETX/Bacillus mosquitocidal toxin MTX2 APG02742.0 40 PF14200Ricin-type beta-trefoil lectin 50 147 domain-like PF05431 InsecticidalCrystal Toxin, P42 157 353 APG02742.1 41 Alternate start PF14200Ricin-type beta-trefoil lectin 53 143 domain-like PF05431 InsecticidalCrystal Toxin, P42 153 349 APG03055.0 42 PF03318 Clostridium epsilontoxin 91 257 ETX/Bacillus mosquitocidal toxin MTX2 APG03055.1 43Alternate start PF03318 Clostridium epsilon toxin 86 252 ETX/Bacillusmosquitocidal toxin MTX2 APG03055.2 44 Signal peptide PF03318Clostridium epsilon toxin 48 215 removed ETX/Bacillus mosquitocidaltoxin MTX2 APG03101.0 45 PF03318 Clostridium epsilon toxin 103 301ETX/Bacillus mosquitocidal toxin MTX2 APG03101.1 46 Alternate startPF03318 Clostridium epsilon toxin 67 265 ETX/Bacillus mosquitocidaltoxin MTX2 APG03147.0 47 PF14200 Ricin-type beta-trefoil lectin 9 97domain-like PF05431 Insecticidal Crystal Toxin, P42 159 348 APG03187.048 PF05431 Insecticidal Crystal Toxin, P42 155 356 APG03187.1 49Alternate start PF05431 Insecticidal Crystal Toxin, P42 155 356APG03195.0 50 no PFAM domains APG03195.1 51 Alternate start no PFAMdomains APG03195.2 52 Signal peptide no PFAM removed domains APG03279.053 PF00652 Ricin-type beta-trefoil lectin 44 158 domain PF05431Insecticidal Crystal Toxin, P42 171 364 APG03324.0 54 PF03318Clostridium epsilon toxin 105 314 ETX/Bacillus mosquitocidal toxin MTX2APG03324.1 55 Alternate start PF03318 Clostridium epsilon toxin 100 309ETX/Bacillus mosquitocidal toxin MTX2 APG03498.0 56 PF03945 deltaendotoxin, N-terminal 79 290 domain PF00555 delta endotoxin 297 482PF03944 delta endotoxin 510 655 APG03498.1 57 3′ Truncation PF03945delta endotoxin, N-terminal 78 290 domain PF00555 delta endotoxin 297482 PF03944 delta endotoxin 510 655 APG03889.0 58 PF03318 Clostridiumepsilon toxin 123 320 ETX/Bacillus mosquitocidal toxin MTX2 APG03889.159 Signal peptide PF03318 Clostridium epsilon toxin 90 287 removedETX/Bacillus mosquitocidal toxin MTX2 APG03900.0 60 PF03318 Clostridiumepsilon toxin 81 297 ETX/Bacillus mosquitocidal toxin MTX2 APG04069.0 61PF07691 PA14 domain 45 170 PF03495 Clostridial binary toxin 196 601B/anthrax toxin PA APG04069.1 62 Signal peptide PF07691 PA14 domain 18143 removed PF03495 Clostridial binary toxin 169 574 B/anthrax toxin PAAPG04345.0 63 PF03318 Clostridium epsilon toxin 99 253 ETX/Bacillusmosquitocidal toxin MTX2 APG04345.1 64 Alternate start PF03318Clostridium epsilon toxin 78 233 ETX/Bacillus mosquitocidal toxin MTX2APG04415.0 65 PF03945 delta endotoxin, N-terminal 43 275 domain PF00555delta endotoxin 283 474 PF03944 delta endotoxin 484 621 APG04586.0 66PF03318 Clostridium epsilon toxin 103 287 ETX/Bacillus mosquitocidaltoxin MTX2 APG04671.0 67 PF03945 delta endotoxin, N-terminal 62 288domain PF00555 delta endotoxin 294 505 PF03944 delta endotoxin 516 647APG04671.1 68 Alternate start PF03945 delta endotoxin, N-terminal 59 285domain PF00555 delta endotoxin 291 502 PF03944 delta endotoxin 513 644APG04671 69 no PFAM Split-Cry C- domains terminus (APG03815.0)APG04699.0 70 PF03318 Clostridium epsilon toxin 43 247 ETX/Bacillusmosquitocidal toxin MTX2 APG04912.0 71 PF00388Phosphatidylinositol-specific 57 206 phospholipase C, X domain PF14200Ricin-type beta-trefoil lectin 516 616 domain-like APG05259.0 72 PF05431Insecticidal Crystal Toxin, P42 174 379 APG05259.1 73 Alternate startPF05431 Insecticidal Crystal Toxin, P42 174 379 APG05284.0 74 PF00652Ricin-type beta-trefoil lectin 10 143 domain PF05431 InsecticidalCrystal Toxin, P42 161 341 APG05311.0 75 PF03945 delta endotoxin,N-terminal 147 389 domain PF00555 delta endotoxin 395 581 PF03944 deltaendotoxin 591 729 APG05311.1 76 Alternate start PF03945 delta endotoxin,N-terminal 62 304 and 3′ domain Truncation PF00555 delta endotoxin 310496 PF03944 delta endotoxin 506 644 APG05341.0 77 PF03318 Clostridiumepsilon toxin 157 353 ETX/Bacillus mosquitocidal toxin MTX2 APG05341.178 Signal peptide PF03318 Clostridium epsilon toxin 112 308 removedETX/Bacillus mosquitocidal toxin MTX2 APG05669.0 79 PF01823 MAC/Perforindomain 106 301 APG05886.0 80 PF03945 delta endotoxin, N-terminal 79 328domain PF03944 delta endotoxin 555 708 APG05886.1 81 3′ TruncationPF03945 delta endotoxin, N-terminal 79 328 domain PF03944 deltaendotoxin 555 707 APG05896.0 82 PF03318 Clostridium epsilon toxin 68 173ETX/Bacillus mosquitocidal toxin MTX2 APG06043.0 83 PF00388Phosphatidylinositol-specific 68 209 phospholipase C, X domainAPG06043.1 84 Signal peptide PF00388 Phosphatidylinositol-specific 34175 removed phospholipase C, X domain APG06187.0 85 PF01117 Aerolysintoxin 114 247 APG06187.1 86 Signal peptide PF01117 Aerolysin toxin 88221 removed APG06228.0 87 PF03318 Clostridium epsilon toxin 85 288ETX/Bacillus mosquitocidal toxin MTX2 APG06228.1 88 Signal peptidePF03318 Clostridium epsilon toxin 52 255 removed ETX/Bacillusmosquitocidal toxin MTX2 APG06242.0 89 PF03318 Clostridium epsilon toxin140 232 ETX/Bacillus mosquitocidal toxin MTX2 APG06242.1 90 Alternatestart PF03318 Clostridium epsilon toxin 132 224 ETX/Bacillusmosquitocidal toxin MTX2 APG06242.2 91 Signal peptide PF03318Clostridium epsilon toxin 101 193 removed ETX/Bacillus mosquitocidaltoxin MTX2 APG06401.0 92 PF05431 Insecticidal Crystal Toxin, P42 246 446APG06401.1 93 Alternate start PF05431 Insecticidal Crystal Toxin, P42243 443 APG06560.0 94 PF03318 Clostridium epsilon toxin 89 309ETX/Bacillus mosquitocidal toxin MTX2 APG06560.1 95 Signal peptidePF03318 Clostridium epsilon toxin 58 278 removed ETX/Bacillusmosquitocidal toxin MTX2 APG06730.0 96 PF03945 delta endotoxin,N-terminal 35 222 domain PF01473 Putative cell wall binding 323 337repeat PF01473 Putative cell wall binding 344 356 repeat APG06730.1 97Alternate start PF03945 delta endotoxin, N-terminal 7 195 domain PF01473Putative cell wall binding 296 310 repeat PF01473 Putative cell wallbinding 317 329 repeat APG06801.0 98 PF05431 Insecticidal Crystal Toxin,P42 184 249 APG06801.1 99 Alternate start PF05431 Insecticidal CrystalToxin, P42 184 249 APG06963.0 100 PF05791 Bacillus haemolytic 73 208enterotoxin (HBL) APG06971.0 101 PF01338 Bacillus thuringiensis toxin 19215 APG06971.1 102 Alternate start PF01338 Bacillus thuringiensis toxin19 215 APG07083.0 103 PF03318 Clostridium epsilon toxin 88 256ETX/Bacillus mosquitocidal toxin MTX2 APG07125.0 104 PF03945 deltaendotoxin, N-terminal 7 160 domain APG07125.1 105 Alternate startPF03945 delta endotoxin, N-terminal 1 130 domain APG07235.0 106 no PFAMdomains APG07235.1 107 Signal peptide no PFAM removed domains APG07247.0108 PF05431 Insecticidal Crystal Toxin, P42 196 389 APG07247.1 109Signal peptide PF05431 Insecticidal Crystal Toxin, P42 169 362 removedAPG07334.0 110 PF05431 Insecticidal Crystal Toxin, P42 200 389APG07334.1 111 PF05431 Insecticidal Crystal Toxin, P42 171 360APG07383.0 112 PF03945 delta endotoxin, N-terminal 68 316 domain PF00555delta endotoxin 325 398 PF03944 delta endotoxin 525 658 APG07383.1 113Alternate start PF03945 delta endotoxin, N-terminal 45 293 domainPF00555 delta endotoxin 302 375 PF03944 delta endotoxin 502 635APG07491.0 114 PF05431 Insecticidal Crystal Toxin, P42 199 389APG07491.1 115 Signal peptide PF05431 Insecticidal Crystal Toxin, P42163 353 removed APG07526.0 116 PF07691 PA14 domain 49 175 PF03495Clostridial binary toxin 215 628 B/anthrax toxin PA PF09259 Fungalimmunomodulatory 866 950 protein Fve APG07526.1 117 Signal peptidePF07691 PA14 domain 22 148 removed PF03495 Clostridial binary toxin 188601 B/anthrax toxin PA PF09259 Fungal immunomodulatory 839 923 proteinFve APG07606.0 118 PF03318 Clostridium epsilon toxin 151 296ETX/Bacillus mosquitocidal toxin MTX2 APG07606.1 119 Alternate startPF03318 Clostridium epsilon toxin 112 257 ETX/Bacillus mosquitocidaltoxin MTX2 APG07606.2 120 Signal peptide PF03318 Clostridium epsilontoxin 81 226 removed ETX/Bacillus mosquitocidal toxin MTX2 APG07679.0121 PF03945 delta endotoxin, N-terminal 94 352 domain PF00555 deltaendotoxin 362 541 PF03944 delta endotoxin 559 699 APG07679.1 122 3′Truncation PF03945 delta endotoxin, N-terminal 94 352 domain PF00555delta endotoxin 362 541 PF03944 delta endotoxin 559 698 APG07799.0 123PF03945 delta endotoxin, N-terminal 99 310 domain PF03944 deltaendotoxin 542 695 APG07799 124 PF07029 CryBP1 protein 38 190 Split-CryC-terminus (APG07295.0) APG07868.0 125 PF05431 Insecticidal CrystalToxin, P42 188 379 APG07889.0 126 PF03945 delta endotoxin, N-terminal 69311 domain PF00555 delta endotoxin 317 503 PF03944 delta endotoxin 513655 APG07889.1 127 3′ Truncation PF03945 delta endotoxin, N-terminal 69311 domain PF00555 delta endotoxin 317 503 PF03944 delta endotoxin 513654 APG08005.0 128 PF00652 Ricin-type beta-trefoil lectin 69 184 domainPF05431 Insecticidal Crystal Toxin, P42 196 393 APG08005.1 129 Alternatestart PF00652 Ricin-type beta-trefoil lectin 45 160 domain PF05431Insecticidal Crystal Toxin, P42 172 369 APG08195.0 130 PF05431Insecticidal Crystal Toxin, P42 188 379 APG08330.0 131 no PFAM domainsAPG08330.1 132 Alternate start no PFAM domains APG08361.0 133 PF00652Ricin-type beta-trefoil lectin 69 184 domain PF05431 InsecticidalCrystal Toxin, P42 196 393 APG08361.1 134 Alternate start PF00652Ricin-type beta-trefoil lectin 45 160 domain PF05431 InsecticidalCrystal Toxin, P42 172 369 APG08369.0 135 PF03318 Clostridium epsilontoxin 89 298 ETX/Bacillus mosquitocidal toxin MTX2 APG08369.1 136 Signalpeptide PF03318 Clostridium epsilon toxin 56 265 removed ETX/Bacillusmosquitocidal toxin MTX2 APG08430.0 137 PF03945 delta endotoxin,N-terminal 67 286 domain PF00555 delta endotoxin 300 505 PF03944 deltaendotoxin 516 660 APG08452.0 138 PF03318 Clostridium epsilon toxin 83246 ETX/Bacillus mosquitocidal toxin MTX2 APG08835.0 139 PF03945 deltaendotoxin, N-terminal 68 342 domain PF03944 delta endotoxin 567 709APG08835.1 140 3′ Truncation PF03945 delta endotoxin, N-terminal 68 342domain PF03944 delta endotoxin 567 708 APG08992.0 141 PF07691 PA14domain 80 172 PF03495 Clostridial binary toxin 203 610 B/anthrax toxinPA APG08992.1 142 Signal peptide PF07691 PA14 domain 53 145 removedPF03495 Clostridial binary toxin 176 583 B/anthrax toxin PA APG09100.0143 PF03318 Clostridium epsilon toxin 133 329 ETX/Bacillus mosquitocidaltoxin MTX2 APG09100.1 144 Signal peptide PF03318 Clostridium epsilontoxin 106 302 removed ETX/Bacillus mosquitocidal toxin MTX2 APG09155.0145 PF03318 Clostridium epsilon toxin 86 285 ETX/Bacillus mosquitocidaltoxin MTX2 APG09155.1 146 Signal peptide PF03318 Clostridium epsilontoxin 53 252 removed ETX/Bacillus mosquitocidal toxin MTX2 APG09211.0147 PF03318 Clostridium epsilon toxin 171 278 ETX/Bacillus mosquitocidaltoxin MTX2 APG09211.1 148 Signal peptide PF03318 Clostridium epsilontoxin 130 237 removed ETX/Bacillus mosquitocidal toxin MTX2 APG09338.0149 PF03318 Clostridium epsilon toxin 159 251 ETX/Bacillus mosquitocidaltoxin MTX2 APG09338.1 150 Signal peptide PF03318 Clostridium epsilontoxin 131 223 removed ETX/Bacillus mosquitocidal toxin MTX2 APG09441.0151 PF03945 delta endotoxin, N-terminal 161 336 domain PF01473 Putativecell wall binding 521 532 repeat PF01473 Putative cell wall binding 564578 repeat APG09441.1 152 Alternate start PF03945 delta endotoxin,N-terminal 124 299 domain PF01473 Putative cell wall binding 484 495repeat PF01473 Putative cell wall binding 527 541 repeat APG09459.0 153PF03318 Clostridium epsilon toxin 176 340 ETX/Bacillus mosquitocidaltoxin MTX2 APG09459.1 154 Alternate start PF03318 Clostridium epsilontoxin 166 330 ETX/Bacillus mosquitocidal toxin MTX2 APG09459.2 155Signal peptide PF03318 Clostridium epsilon toxin 136 300 removedETX/Bacillus mosquitocidal toxin MTX2 APG09460.0 156 PF03318 Clostridiumepsilon toxin 111 258 ETX/Bacillus mosquitocidal toxin MTX2 APG09460.1157 Signal peptide PF03318 Clostridium epsilon toxin 80 228 removedETX/Bacillus mosquitocidal toxin MTX2 APG09492.0 158 PF03318 Clostridiumepsilon toxin 95 295 ETX/Bacillus mosquitocidal toxin MTX2 APG09492.1159 Signal peptide PF03318 Clostridium epsilon toxin 62 262 removedETX/Bacillus mosquitocidal toxin MTX2 APG09604.0 160 PF03945 deltaendotoxin, N-terminal 85 311 domain PF00555 delta endotoxin 318 545PF03944 delta endotoxin 556 694 APG09604.1 161 3′ Truncation PF03945delta endotoxin, N-terminal 85 311 domain PF00555 delta endotoxin 318545 PF03944 delta endotoxin 556 694 APG09768.0 162 PF05431 InsecticidalCrystal Toxin, P42 199 392 APG09768.1 163 Alternate start PF05431Insecticidal Crystal Toxin, P42 165 358 APG09869.0 164 PF03318Clostridium epsilon toxin 102 316 ETX/Bacillus mosquitocidal toxin MTX2APG09869.1 165 Alternate start PF03318 Clostridium epsilon toxin 72 289ETX/Bacillus mosquitocidal toxin MTX2 APG09869.2 166 Signal peptidePF03318 Clostridium epsilon toxin 51 263 removed ETX/Bacillusmosquitocidal toxin MTX2 APG09953.0 167 PF03318 Clostridium epsilontoxin 177 245 ETX/Bacillus mosquitocidal toxin MTX2 APG09953.1 168Signal peptide PF03318 Clostridium epsilon toxin 148 216 removedETX/Bacillus mosquitocidal toxin MTX2 APG09977.0 169 PF05431Insecticidal Crystal Toxin, P42 188 377 APG09977.1 170 Signal peptidePF05431 Insecticidal Crystal Toxin, P42 161 350 removed APG00765.0 171PF03318 Clostridium epsilon toxin 108 317 ETX/Bacillus mosquitocidaltoxin MTX2 APG00765.1 172 Alternate start PF03318 Clostridium epsilontoxin 80 289 ETX/Bacillus mosquitocidal toxin MTX2 APG00765.2 173 Signalpeptide PF03318 Clostridium epsilon toxin 54 263 removed ETX/Bacillusmosquitocidal toxin MTX2 APG01166.0 174 PF05431 Insecticidal CrystalToxin, P42 212 406 APG01166.1 175 Alternate start PF05431 InsecticidalCrystal Toxin, P42 206 400 APG01166.2 176 Signal peptide PF05431Insecticidal Crystal Toxin, P42 172 366 removed APG01488.0 177 PF03318Clostridium epsilon toxin 93 254 ETX/Bacillus mosquitocidal toxin MTX2APG03067.0 178 PF03318 Clostridium epsilon toxin 123 320 ETX/Bacillusmosquitocidal toxin MTX2 APG03067.1 179 Signal peptide PF03318Clostridium epsilon toxin 90 287 removed ETX/Bacillus mosquitocidaltoxin MTX2 APG04947.0 180 PF03318 Clostridium epsilon toxin 91 284ETX/Bacillus mosquitocidal toxin MTX2 APG04947.1 181 Alternate startPF03318 Clostridium epsilon toxin 69 262 ETX/Bacillus mosquitocidaltoxin MTX2 APG05791.0 182 PF03318 Clostridium epsilon toxin 123 320ETX/Bacillus mosquitocidal toxin MTX2 APG05791.1 183 Signal peptidePF03318 Clostridium epsilon toxin 90 287 removed ETX/Bacillusmosquitocidal toxin MTX2 APG05956.0 184 PF03318 Clostridium epsilontoxin 97 272 ETX/Bacillus mosquitocidal toxin MTX2 APG05956.1 185 Signalpeptide PF03318 Clostridium epsilon toxin 66 242 removed ETX/Bacillusmosquitocidal toxin MTX2 APG06419.0 186 PF03945 delta endotoxin,N-terminal 85 311 domain PF00555 delta endotoxin 318 549 PF03944 deltaendotoxin 560 698 APG06419.1 187 3′ Truncation PF03945 delta endotoxin,N-terminal 85 311 domain PF00555 delta endotoxin 318 430 PF03944 deltaendotoxin 560 698 APG06727.0 188 PF03318 Clostridium epsilon toxin 122331 ETX/Bacillus mosquitocidal toxin MTX2 APG06727.1 189 Alternate startPF03318 Clostridium epsilon toxin 80 289 ETX/Bacillus mosquitocidaltoxin MTX2 APG06727.2 190 Signal peptide PF03318 Clostridium epsilontoxin 54 263 removed ETX/Bacillus mosquitocidal toxin MTX2 APG03898.0194 PF03318 Clostridium epsilon toxin 101 310 ETX/Bacillus mosquitocidaltoxin MTX2 APG03898.1 195 Alternate start PF03318 Clostridium epsilontoxin 80 289 ETX/Bacillus mosquitocidal toxin MTX2 APG03898.2 196 Signalpeptide PF03318 Clostridium epsilon toxin 54 263 removed ETX/Bacillusmosquitocidal toxin MTX2 APG06237.0 197 PF05431 Insecticidal CrystalToxin, P42 218 379 APG04021.0 198 PF03318 Clostridium epsilon toxin 131330 ETX/Bacillus mosquitocidal toxin MTX2 APG04021.1 199 Alternate startPF03318 Clostridium epsilon toxin 131 330 ETX/Bacillus mosquitocidaltoxin MTX2 APG04021.2 200 Signal peptide PF03318 Clostridium epsilontoxin 104 303 removed ETX/Bacillus mosquitocidal toxin MTX2 APG04627.0201 PF05431 Insecticidal Crystal Toxin, P42 218 379 APG05411.0 202PF14200 Ricin-type beta-trefoil lectin 72 149 domain-like PF05431Insecticidal Crystal Toxin, P42 230 390 APG05411.1 203 Signal peptidePF14200 Ricin-type beta-trefoil lectin 45 122 removed domain-likePF05431 Insecticidal Crystal Toxin, P42 203 363 APG08768.0 204 PF03318Clostridium epsilon toxin 108 311 ETX/Bacillus mosquitocidal toxin MTX2APG08768.1 205 Alternate start PF03318 Clostridium epsilon toxin 103 306ETX/Bacillus mosquitocidal toxin MTX2

Recombinant or synthetic nucleic acids encoding the pesticidalpolypeptides disclosed herein are also provided. Of particular interestare nucleic acid sequences that have been designed for expression in aplant of interest. That is, the nucleic acid sequence can be optimizedfor increased expression in a host plant. A pesticidal protein of theinvention can be back-translated to produce a nucleic acid comprisingcodons optimized for expression in a particular host, for example, acrop plant. In another embodiment, the polynucleotides encoding thepolypeptides provided herein may be optimized for increased expressionin the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference. Expression of such acoding sequence by the transformed plant (e.g., dicot or monocot) willresult in the production of a pesticidal polypeptide and conferincreased resistance in the plant to a pest. Recombinant and syntheticnucleic acid molecules encoding the pesticidal proteins of the inventiondo not include the naturally occurring bacterial sequence encoding theprotein.

A “recombinant polynucleotide” or “recombinant nucleic acid” comprises acombination of two or more chemically linked nucleic acid segments whichare not found directly joined in nature. By “directly joined” isintended the two nucleic acid segments are immediately adjacent andjoined to one another by a chemical linkage. In specific embodiments,the recombinant polynucleotide comprises a polynucleotide of interest ora variant or fragment thereof such that an additional chemically linkednucleic acid segment is located either 5′, 3′ or internal to thepolynucleotide of interest. Alternatively, the chemically-linked nucleicacid segment of the recombinant polynucleotide can be formed by deletionof a sequence. The additional chemically linked nucleic acid segment orthe sequence deleted to join the linked nucleic acid segments can be ofany length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20or greater nucleotides. Various methods for making such recombinantpolynucleotides include chemical synthesis or by the manipulation ofisolated segments of polynucleotides by genetic engineering techniques.In specific embodiments, the recombinant polynucleotide can comprise arecombinant DNA sequence or a recombinant RNA sequence. A “fragment of arecombinant polynucleotide or nucleic acid” comprises at least one of acombination of two or more chemically linked amino acid segments whichare not found directly joined in nature.

Fragments of a polynucleotide (RNA or DNA) may encode protein fragmentsthat retain activity. In specific embodiments, a fragment of arecombinant polynucleotide or a recombinant polynucleotide constructcomprises at least one junction of the two or more chemically linked oroperably linked nucleic acid segments which are not found directlyjoined in nature. A fragment of a polynucleotide that encodes abiologically active portion of a polypeptide that retains pesticidalactivity will encode at least 25, 30, 40, 50, 60, 70, 75, 80, 90, 100,110, 120, 125, 130, 140, 150, 160, 170, 175, 180, contiguous aminoacids, or up to the total number of amino acids present in a full-lengthpolypeptide as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205. In specific embodiments, suchpolypeptide fragments are active fragment, and in still otherembodiments, the polypeptide fragment comprises a recombinantpolypeptide fragment. As used herein, a fragment of a recombinantpolypeptide comprises at least one of a combination of two or morechemically linked amino acid segments which are not found directlyjoined in nature.

By “Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a deletion and/or addition of oneor more nucleotides at one or more internal sites within the nativepolynucleotide and/or a substitution of one or more nucleotides at oneor more sites in the native polynucleotide. As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotidesequence or amino acid sequence, respectively.

Variants of a particular polynucleotide of the invention (i.e., thereference polynucleotide) can also be evaluated by comparison of thepercent sequence identity between the polypeptide encoded by a variantpolynucleotide and the polypeptide encoded by the referencepolynucleotide. Thus, for example, an isolated polynucleotide thatencodes a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173,174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201,202, 203, 204, and/or 205 are disclosed. Percent sequence identitybetween any two polypeptides can be calculated using sequence alignmentprograms and parameters described elsewhere herein. Where any given pairof polynucleotides of the invention is evaluated by comparison of thepercent sequence identity shared by the two polypeptides they encode,the percent sequence identity between the two encoded polypeptides is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, and/or 205. In other embodiments, the variantof the polynucleotide provided herein differs from the native sequenceby at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.

Variant polynucleotide and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more different pesticidalprotein disclosed herein (SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205) is manipulated to create a newpesticidal protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between the pesticidialsequences provided herein and other known pesticidial genes to obtain anew gene coding for a protein with an improved property of interest,such as an increased K_(m) in the case of an enzyme. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458. A “shuffled” nucleic acid isa nucleic acid produced by a shuffling procedure such as any shufflingprocedure set forth herein. Shuffled nucleic acids are produced byrecombining (physically or virtually) two or more nucleic acids (orcharacter strings), for example in an artificial, and optionallyrecursive, fashion. Generally, one or more screening steps are used inshuffling processes to identify nucleic acids of interest; thisscreening step can be performed before or after any recombination step.In some (but not all) shuffling embodiments, it is desirable to performmultiple rounds of recombination prior to selection to increase thediversity of the pool to be screened. The overall process ofrecombination and selection are optionally repeated recursively.Depending on context, shuffling can refer to an overall process ofrecombination and selection, or, alternately, can simply refer to therecombinational portions of the overall process.

In one embodiments, a method of obtaining a polynucleotide that encodesan improved polypeptide comprising pesticidal activity is provided,wherein the improved polypeptide has at least one improved property overany one of SEQ ID NOS: 1-205. Such methods can comprises (a) recombininga plurality of parental polynucleotides to produce a library ofrecombinant polynucleotides encoding recombinant pesticidalpolypeptides; (b) screening the library to identify a recombinantpolynucleotide that encodes an improved recombinant pesticidalpolypeptide that has an enhanced property improved over the parentalpolynucleotide; (c) recovering the recombinant polynucleotide thatencodes the improved recombinant pesticidal polypeptide identified in(b); and, (d) repeating steps (a), (b) and (c) using the recombinantpolynucleotide recovered in step (c) as one of the plurality of parentalpolynucleotides in repeated step (a).

iii. Sequence Comparisons

As used herein, the term “identity” or “percent identity” when used withrespect to a particular pair of aligned amino acid sequences, refers tothe percent amino acid sequence identity that is obtained by countingthe number of identical matches in the alignment and dividing suchnumber of identical matches by the length of the aligned sequences. Asused herein, the term “similarity” or “percent similarity” when usedwith respect to a particular pair of aligned amino acid sequences,refers to the sum of the scores that are obtained from a scoring matrixfor each amino acid pair in the alignment divided by the length of thealigned sequences.

Unless otherwise stated, identity and similarity will be calculated bythe Needleman-Wunsch global alignment and scoring algorithms (Needlemanand Wunsch (1970) J. Mol. Biol. 48(3):443-453) as implemented by the“needle” program, distributed as part of the EMBOSS software package(Rice, P. Longden, I. and Bleasby, A., EMBOSS: The European MolecularBiology Open Software Suite, 2000, Trends in Genetics 16, (6) pp276-277, versions 6.3.1 available from EMBnet atembnet.org/resource/emboss and emboss.sourceforge.net, among othersources) using default gap penalties and scoring matrices (EBLOSUM62 forprotein and EDNAFULL for DNA). Equivalent programs may also be used. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide residue matches and an identical percent sequenceidentity when compared to the corresponding alignment generated byneedle from EMBOSS version 6.3.1.

Additional mathematical algorithms are known in the art and can beutilized for the comparison of two sequences. See, for example, thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTprograms of Altschul et al. (1990) J. Mol. Biol. 215:403. BLASTnucleotide searches can be performed with the BLASTN program (nucleotidequery searched against nucleotide sequences) to obtain nucleotidesequences homologous to pesticidal-like nucleic acid molecules of theinvention, or with the BLASTX program (translated nucleotide querysearched against protein sequences) to obtain protein sequenceshomologous to pesticidal nucleic acid molecules of the invention. BLASTprotein searches can be performed with the BLASTP program (protein querysearched against protein sequences) to obtain amino acid sequenceshomologous to pesticidal protein molecules of the invention, or with theTBLASTN program (protein query searched against translated nucleotidesequences) to obtain nucleotide sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacid substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art and described, e.g., inDayhoff et al. (1978) “A model of evolutionary change in proteins.” In“Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O.Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. andHenikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. TheBLOSUM62 matrix is often used as a default scoring substitution matrixin sequence alignment protocols. The gap existence penalty is imposedfor the introduction of a single amino acid gap in one of the alignedsequences, and the gap extension penalty is imposed for each additionalempty amino acid position inserted into an already opened gap. Thealignment is defined by the amino acids positions of each sequence atwhich the alignment begins and ends, and optionally by the insertion ofa gap or multiple gaps in one or both sequences, so as to arrive at thehighest possible score. While optimal alignment and scoring can beaccomplished manually, the process is facilitated by the use of acomputer-implemented alignment algorithm, e.g., gapped BLAST 2.0,described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, andmade available to the public at the National Center for BiotechnologyInformation Website (www.ncbi.nlm.nih.gov). Optimal alignments,including multiple alignments, can be prepared using, e.g., PSI-BLAST,available through www.ncbi.nlm.nih.gov and described by Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402.

With respect to an amino acid sequence that is optimally aligned with areference sequence, an amino acid residue “corresponds to” the positionin the reference sequence with which the residue is paired in thealignment. The “position” is denoted by a number that sequentiallyidentifies each amino acid in the reference sequence based on itsposition relative to the N-terminus. For example, in SEQ ID NO: 1position 1 is M, position 2 is E, position 3 is L, etc. When a testsequence is optimally aligned with SEQ ID NO: 1, a residue in the testsequence that aligns with the L at position 3 is said to “correspond toposition 3” of SEQ ID NO: 1. Owing to deletions, insertion, truncations,fusions, etc., that must be taken into account when determining anoptimal alignment, in general the amino acid residue number in a testsequence as determined by simply counting from the N-terminal will notnecessarily be the same as the number of its corresponding position inthe reference sequence. For example, in a case where there is a deletionin an aligned test sequence, there will be no amino acid thatcorresponds to a position in the reference sequence at the site ofdeletion. Where there is an insertion in an aligned reference sequence,that insertion will not correspond to any amino acid position in thereference sequence. In the case of truncations or fusions there can bestretches of amino acids in either the reference or aligned sequencethat do not correspond to any amino acid in the corresponding sequence.

iv. Antibodies

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; and U.S. Pat. No. 4,196,265). These antibodiescan be used in kits for the detection and isolation of toxinpolypeptides. Thus, this disclosure provides kits comprising antibodiesthat specifically bind to the polypeptides described herein, including,for example, polypeptides having the sequence of SEQ ID NOs: 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and/or 205.

II. Pests

The compositions and methods provided herein are useful against avariety of pests. “Pests” includes but is not limited to, insects,fungi, bacteria, nematodes, acarids, protozoan pathogens,animal-parasitic liver flukes, and the like. Pests of particularinterest are insect pests, particularly insect pests that causesignificant damage to agricultural plants. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, or nematodes. Innon-limiting embodiments, the insect pest comprises Western cornrootworm, Diabrotica virgifera virgifera; Fall armyworm, Spodopterafrupperda; Colorado potato beetle, Leptinotarsa decemlineata; Cornearworm, Helicoverpa zea (in North America same species attacks cottonand called cotton bollworm); European corn borer, Ostrinia nubilalis;Black cutworm, Agrotis ipsilon; Diamondback moth, Plutella xylostella;Velvetbean caterpillar, Anticarsia gemmatalis; Southwestern corn borer,Diatraea grandiosella; Cotton bollworm, Helicoverpa armigera (foundother than USA in rest of the world); Southern green stinkbug, Nezaraviridula; Green stinkbug, Chinavia halaris; Brown marmorated stinkbug,Halyomorpha halys; and Brown stinbug, Euschistus servus, Euschistusheros (Neotropical brown stink bug OR soy stink bug); Piezodorusguildinii (red-banded stink bug); Dichelops melacanthus (no common name)and/or Dichelops furcatus (no common name); an aphid, such as a soybeanaphid. In other embodiments, the pest comprises a nematode including,but not limited to, Meloidogyne hapla (Northern root-knot nematode);Meloidogyne enterolobii, Meloidogyne arenaria (peanut root-knotnematode); and Meloidogyne javanica.

The term “insect pests” as used herein refers to insects and othersimilar pests such as, for example, those of the order Acari including,but not limited to, mites and ticks. Insect pests of the presentinvention include, but are not limited to, insects of the orderLepidoptera, e.g. Achoroia grisella, Acleris gloverana, Acleris variana,Adoxophyes orana, Agrotis ipsilon, Alabama argillacea, Alsophilapometaria, Amyelois transitella, Anagasta kuehniella, Anarsialineatella, Anisota senatoria, Antheraea pernyi, Anticarsia gemmatalis,Archips sp., Argyrotaenia sp., Athetis mindara, Bombyx mori, Bucculatrixthurberiella, Cadra cautella, Choristoneura sp., Cochylls hospes, Coliaseurytheme, Corcyra cephalonica, Cydia latiferreanus, Cydia pomonella,Datana integerrima, Dendrolimus sibericus, Desmiafeneralis, Diaphaniahyalinata, Diaphania nitidalis, Diatraea grandiosella, Diatraeasaccharalis, Ennomos subsignaria, Eoreuma loftini, Esphestia elutella,Erannis tilaria, Estigmene acrea, Eulia salubricola, Eupocoelliaambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea, Euxoamessoria, Galleria mellonella, Grapholita molesta, Harrisina americana,Helicoverpa subflexa, Helicoverpa zea, Heliothis virescens, Hemileucaoliviae, Homoeosoma electellum, Hyphantia cunea, Keiferialycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellarialugubrosa, Leucoma salicis, Lobesia botrana, Loxostege sticticalis,Lymantria dispar, Macalla thyrisalis, Malacosoma sp., Mamestrabrassicae, Mamestra configurata, Manduca quinquemaculata, Manduca sexta,Maruca testulalis, Melanchra picta, Operophtera brumata, Orgyia sp.,Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota stultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp.,Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udearubigalis, Xylomyges curiails, and Yponomeuta padella.

Insect pests also include insects selected from the orders Diptera,Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera,Coleoptera. Insect pests of the invention for the major crops include,but are not limited to: Maize: Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Helicoverpa zeae, corn earworm;Spodoptera frugiperda, fall armyworm; Diatraea grandiosella,southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalkborer; Diatraea saccharalis, surgarcane borer; western corn rootworm,e.g., Diabrotica virgifera virgifera; northern corn rootworm, e.g.,Diabrotica longicornis barberi; southern corn rootworm, e.g., Diabroticaundecimpunctata howardi; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Euschistus heros (Neotropical brown stink bug OR soystink bug); Piezodorus guildinii (red-banded stink bug); Dichelopsmelacanthus (no common name); Dichelops furcatus (no common name);Blissus leucopterus leucopterus, chinch bug; Melanoplus femurrubrum,redlegged grasshopper; Melanoplus sanguinipes, migratory grasshopper;Hylemya platura, seedcorn maggot; Agromyza parvicornis, corn blotchleafminer; Anaphothrips obscrurus, grass thrips; Solenopsis milesta,thief ant; Tetranychus urticae, two spotted spider mite; Sorghum: Chilopartellus, sorghum borer; Spodoptera frugiperda, fall armyworm;Helicoverpa zea, corn earworm; Elasmopalpus lignosellus, leser cornstalkborer; Feltia subterranea, granulate cutworm; Phyllophaga crinita, whitegrub; Eleodes, Conoderus, and Aeolus spp., wireworms; Oulema melanopus,cereal leaf beetle; Chaetocnema pulicaria, corn flea beetle;Sphenophorus maidis, maize billbug; Rhopalosiphum maidis; corn leafaphid; Sipha flava, yellow sugarcane aphid; chinch bug, e.g., Blissusleucopterus leucopterus; Contarinia sorghicola, sorghum midge;Tetranychus cinnabarinus, carmine spider mite; Tetranychus urticae,two-spotted spider mite; Wheat: Pseudaletia unipunctata, army worm;Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus, lessercornstalk borer; Agrotis orthogonia, pale western cutworm; Elasmopalpuslignosellus, lesser cornstalk borer; Oulema melanopus, cereal leafbeetle; Hypera punctata, clover leaf weevil; southern corn rootworm,e.g., Diabrotica undecimpunctata howardi; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Cylindrocupturus adspersus, sunflower stem weevil; Smicronyx fulus, redsunflower seed weevil; Smicronyx sordidus, gray sunflower seed weevil;Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; Zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, tobacco budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; boll weevil, e.g., Anthonomus grandis; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, two-spottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, rice leafhoper;chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternum hilare,green stink bug; Soybean: Pseudoplusia includens, soybean looper;Anticarsia gemmatalis, velvetbean caterpillar; Plathypena scabra, greencloverworm; Ostrinia nubilalis, European corn borer; Agrotis ipsilon,black cutworm; Spodoptera exigua, beet armyworm; Heliothis virescens,tobacco budworm; Helicoverpa zea, cotton bollworm; Epilachna varivestis,Mexican bean beetle; Myzus persicae, green peach aphid; Empoasca fabae,potato leafhopper; Acrosternum hilare, green stink bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus differentialis,differential grasshopper; Hylemya platura, seedcorn maggot; Sericothripsvariabilis, soybean thrips; Thrips tabaci, onion thrips; Tetranychusturkestani, strawberry spider mite; Tetranychus urticae, two-spottedspider mite; Barley: Ostrinia nubilalis, European corn borer; Agrotisipsilon, black cutworm; Schizaphis graminum, greenbug; chinch bug, e.g.,Blissus leucopterus leucopterus; Acrosternum hilare, green stink bug;Euschistus servus, brown stink bug; Jylemya platura, seedcorn maggot;Mayetiola destructor, Hessian fly; Petrobia latens, brown wheat mite;Oil Seed Rape: Vrevicoryne brassicae, cabbage aphid; Phyllotretacruciferae, crucifer flea beetle; Phyllotreta striolata, striped fleabeetle; Phyllotreta nemorum, striped turnip flea beetle; Meligethesaeneus, rapeseed beetle; and the pollen beetles Meligethes rufimanus,Meligethes nigrescens, Meligethes canadianus, and Meligethesviridescens; Potato: Leptinotarsa decemlineata, Colorado potato beetle.

The methods and compositions provided herein may be effective againstHemiptera such as Lygus hesperus, Lygus lineolaris, Lygus pratensis,Lygus rugulipennis Popp, Lygus pabulinus, Calocoris norvegicus, Orthopscompestris, Plesiocoris rugicollis, Cyrtopeltis modestus, Cyrtopeltisnotatus, Spanagonicus albofasciatus, Diaphnocoris chlorinonis,Labopidicola allii, Pseudatomoscelis seriatus, Adelphocoris rapidus,Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae, Nysiusraphanus, Euschistus servus, Nezara viridula, Eurygaster, Coreidae,Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, and Cimicidae. Pestsof interest also include Araecerus fasciculatus, coffee bean weevil;Acanthoscelides obtectus, bean weevil; Bruchus rufinanus, broadbeanweevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus, Mexican beanweevil; Diabrotica balteata, banded cucumber beetle; Cerotomatrifurcata, bean leaf beetle; Diabrotica virgifera, Mexican cornrootworm; Epitrix cucumeris, potato flea beetle; Chaetocnema confinis,sweet potato flea beetle; Hypera postica, alfalfa weevil; Anthonomusquadrigibbus, apple curculio; Sternechus paludatus, bean stalk weevil;Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilus granaries,granary weevil; Craponius inaequalis, grape curculio; Sitophiluszeamais, maize weevil; Conotrachelus nenuphar, plum curculio; Euscepespostfaciatus, West Indian sweet potato weevil; Maladera castanea,Asiatic garden beetle; Rhizotrogus majalis, European chafer;Macrodactylus subspinosus, rose chafer; Tribolium confusum, confusedflour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, redflour beetle; Tenebrio molitor, yellow mealworm.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Insect pests may be tested for pesticidal activity of compositions ofthe invention in early developmental stages, e.g., as larvae or otherimmature forms. The insects may be reared in total darkness at fromabout 20.degree. C. to about 30.degree. C. and from about 30% to about70% relative humidity. Bioassays may be performed as described in Czaplaand Lang (1990) J. Econ. Entomol. 83 (6): 2480-2485. See, also theexperimental section herein.

III. Expression Cassettes

Polynucleotides encoding the pesticidal proteins provided herein can beprovided in expression cassettes for expression in an organism ofinterest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a polynucleotide encoding a pesticidal polypeptideprovided herein that allows for expression of the polynucleotide. Thecassette may additionally contain at least one additional gene orgenetic element to be cotransformed into the organism. Where additionalgenes or elements are included, the components are operably linked.Alternatively, the additional gene(s) or element(s) can be provided onmultiple expression cassettes. Such an expression cassette is providedwith a plurality of restriction sites and/or recombination sites forinsertion of the polynucleotides to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain a selectable marker gene.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a pesticidal polynucleotide of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism of interest, i.e., a plant orbacteria. The promoters of the invention are capable of directing ordriving expression of a coding sequence in a host cell. The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) may be endogenous or heterologous tothe host cell or to each other. As used herein, “heterologous” inreference to a sequence is a sequence that originates from a foreignspecies, or, if from the same species, is substantially modified fromits native form in composition and/or genomic locus by deliberate humanintervention. As used herein, a chimeric gene comprises a codingsequence operably linked to a transcription initiation region that isheterologous to the coding sequence.

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Additional regulatory signals include, but are not limited to,transcriptional initiation start sites, operators, activators,enhancers, other regulatory elements, ribosomal binding sites, aninitiation codon, termination signals, and the like. See, for example,U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrook et al.(1992) Molecular Cloning: A Laboratory Manual, ed. Maniatis et al. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), hereinafter“Sambrook 11”; Davis et al., eds. (1980) Advanced Bacterial Genetics(Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and thereferences cited therein.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, inducible, tissue-preferred, orother promoters for expression in the organism of interest. See, forexample, promoters set forth in WO 99/43838 and in U.S. Pat. Nos:8,575,425; 7,790,846; 8,147,856; 8,586832; 7,772,369; 7,534,939;6,072,050; 5,659,026; 5,608,149; 5,608,144; 5,604,121; 5,569,597;5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; hereinincorporated by reference.

For expression in plants, constitutive promoters also include CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730). Induciblepromoters include those that drive expression of pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen. See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol.89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon(1985) Plant Mol. Virol. 4:111-116; and WO 99/43819, herein incorporatedby reference. Promoters that are expressed locally at or near the siteof pathogen infection may also be used (Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977; Chen et al.(1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci.USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz etal. (1989) Plant Cell 1:961-968; Cordero et al. (1992) Physiol. Mol.Plant Path. 41:189-200; U.S. Pat. No. 5,750,386 (nematode-inducible);and the references cited therein).

Wound-inducible promoters may be used in the constructions of theinvention. Such wound-inducible promoters include pin II promoter (Ryan(1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) NatureBiotechnology 14:494-498); wun1 and wun2 (U.S. Pat. No. 5,428,148); win1and win2 (Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin(McGurl et al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al.(1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters323:73-76); MPI gene (Corderok et al. (1994) Plant 6(2):141-150); andthe like, herein incorporated by reference.

Tissue-preferred promoters for use in the invention include those setforth in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al.(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. GenGenet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505.

Leaf-preferred promoters include those set forth in Yamamoto et al.(1997) Plant J. 12(2):255-265; Kwon et al. (1994) Plant Physiol.105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778;Gotor et al. (1993) Plant 3:509-18; Orozco et al. (1993) Plant Mol.Biol. 23(6):1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci.USA 90(20):9586-9590.

Root-preferred promoters are known and include those in Hire et al.(1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutaminesynthetase gene); Keller and Baumgartner (1991) Plant Cell3(10):1051-1061 (root-specific control element); Sanger et al. (1990)Plant Mol. Biol. 14(3):433-443 (mannopine synthase (MAS) gene ofAgrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22(cytosolic glutamine synthetase (GS)); Bogusz et al. (1990) Plant Cell2(7):633-641; Leach and Aoyagi (1991) Plant Science (Limerick)79(1):69-76 (rolC and rolD); Teeri et al. (1989) EMBO J. 8(2):343-350;Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772 (the VfENOD-GRP3gene promoter); and, Capana et al. (1994) Plant Mol. Biol. 25(4):681-691(rolB promoter). See also U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108. Seed-preferred promoters include, but are not limitedto, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps(myo-inositol-1-phosphate synthase) (see WO 00/11177 and U.S. Pat. No.6,225,529). Gamma-zein is an endosperm-specific promoter. Globulin 1(Glb-1) is a representative embryo-specific promoter. For dicots,seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy,shrunken 1, shrunken 2, Globulin 1, etc. See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed.

For expression in a bacterial host, promoters that function in bacteriaare well-known in the art. Such promoters include any of the knowncrystal protein gene promoters, including the promoters of any of thepesticidal proteins of the invention, and promoters specific for B.thuringiensis sigma factors. Alternatively, mutagenized or recombinantcrystal protein-encoding gene promoters may be recombinantly engineeredand used to promote expression of the novel gene segments disclosedherein.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers areknown and any can be used. See, for example, U.S. Provisionalapplication 62/094,697, filed on Dec. 19, 2014, and U.S. ProvisionalApplication 62/189,505, filed Jul. 7, 2015, both of which are hereinincorporated by reference in their entirety, which discloses glufosinateresistance sequences that can be employed as selectable markers. See,for example, PCT/US2015/066648, filed on Dec. 18, 2015, hereinincorporated by reference in its entirety, which discloses glufosinateresistance sequences that can be employed as selectable markers.

IV. Methods, Host Cells and Plant Cells

As indicated, DNA constructs comprising nucleotide sequences encodingthe pesticidal proteins or active variants or fragment thereof can beused to transform plants of interest or other organisms of interest.Methods for transformation involve introducing a nucleotide constructinto a plant. By “introducing” is intended to introduce the nucleotideconstruct to the plant or other host cell in such a manner that theconstruct gains access to the interior of a cell of the plant or hostcell. The methods of the invention do not require a particular methodfor introducing a nucleotide construct to a plant or host cell, onlythat the nucleotide construct gains access to the interior of at leastone cell of the plant or the host organism. Methods for introducingnucleotide constructs into plants and other host cells are known in theart including, but not limited to, stable transformation methods,transient transformation methods, and virus-mediated methods.

The methods result in a transformed organisms, such as a plant,including whole plants, as well as plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated a polynucleotide encoding at least one pesticidal polypeptideof the invention. It is recognized that other exogenous or endogenousnucleic acid sequences or DNA fragments may also be incorporated intothe plant cell. Agrobacterium-and biolistic-mediated transformationremain the two predominantly employed approaches. However,transformation may be performed by infection, transfection,microinjection, electroporation, microprojection, biolistics or particlebombardment, electroporation, silica/carbon fibers, ultrasound mediated,PEG mediated, calcium phosphate co-precipitation, polycation DMSOtechnique, DEAE dextran procedure, Agro and viral mediated(Caulimoriviruses, Geminiviruses, RNA plant viruses), liposome mediatedand the like.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Methods for transformation are known in the art andinclude those set forth in U.S. Pat. Nos. 8,575,425; 7,692,068;8,802,934; 7,541,517; each of which is herein incorporated by reference.See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858;Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics ofLife Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12;Bates, G. W. (1999) Methods in Molecular Biology 111:359-366; Binns andThomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P.(1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al.(2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski(1995) Plant Physiology 107:1041-1047; Jones et al. (2005) Plant Methods1:5;

Transformation may result in stable or transient incorporation of thenucleic acid into the cell. “Stable transformation” is intended to meanthat the nucleotide construct introduced into a host cell integratesinto the genome of the host cell and is capable of being inherited bythe progeny thereof. “Transient transformation” is intended to mean thata polynucleotide is introduced into the host cell and does not integrateinto the genome of the host cell.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Nail. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent is plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

In specific embodiments, the sequences provide herein can be targeted tospecific cite within the genome of the host cell or plant cell. Suchmethods include, but are not limited to, meganucleases designed againstthe plant genomic sequence of interest (D'Halluin et al. 2013 PlantBiotechnol J); CRISPR-Cas9, TALENs, and other technologies for preciseediting of genomes (Feng, et al. Cell Research 23:1229-1232, 2013,Podevin, et al. Trends Biotechnology, online publication, 2013, Wei etal., J Gen Genomics, 2013, Zhang et al (2013) WO 2013/026740); Cre-loxsite-specific recombination (Dale et al. (1995) Plant J 7:649-659;Lyznik, et al. (2007) Transgenic Plant J 1:1-9; FLP-FRT recombination(Li et al. (2009) Plant Physiol 151:1087-1095); Bxb1-mediatedintegration (Yau et al. Plant J (2011) 701:147-166); zinc-fingermediated integration (Wright et al. (2005) Plant J 44:693-705); Cai etal. (2009) Plant Mol Biol 69:699-709); and homologous recombination(Lieberman-Lazarovich and Levy (2011) Methods Mol Biol 701: 51-65);Puchta (2002) Plant Mol Biol 48:173-182).

The sequence provided herein may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassaya, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.Further provided is a processed plant product or byproduct that retainsthe sequences disclosed herein, including for example, soymeal.

In another embodiment, the genes encoding the pesticidal proteins can beused to transform insect pathogenic organisms. Such organisms includebaculoviruses, fungi, protozoa, bacteria, and nematodes. Microorganismhosts that are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplana) of one or more crops ofinterest may be selected. These microorganisms are selected so as to becapable of successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the gene expressing the pesticidal protein, and desirably, providefor improved protection of the pesticide from environmental degradationand inactivation.

Such microorganisms include archaea, bacteria, algae, and fungi. Ofparticular interest are microorganisms such as bacteria, e.g., Bacillus,Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces,Rhizobium, Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes.Fungi include yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas aeruginosa, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinlandir and phytosphere yeast speciessuch as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces rosues, S. odorus,Kluyveromyces veronae, Aureobasidium pollulans, Bacillus thuringiensis,Escherichia coli, Bacillus subtilis, and the like.

Illustrative prokaryotes, both Gram-negative and gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.Fungi include Phycomycetes and Ascomycetes, e.g., yeast, such asSaccharomyces and Schizosaccharomyces; and Basidiomycetes yeast, such asRhodotorula, Aureobasidium, Sporobolomyces, and the like.

Genes encoding pesticidal proteins can be introduced by means ofelectrotransformation, PEG induced transformation, heat shock,transduction, conjugation, and the like. Specifically, genes encodingthe pesticidal proteins can be cloned into a shuttle vector, forexample, pHT3101 (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218. The shuttle vector pHT3101 containing the coding sequence forthe particular pesticidal protein gene can, for example, be transformedinto the root-colonizing Bacillus by means of electroporation (Lereciuset al. (1989) FEMS Microbiol. Letts. 60: 211-218).

Expression systems can be designed so that pesticidal proteins aresecreted outside the cytoplasm of gram-negative bacteria by fusing anappropriate signal peptide to the amino-terminal end of the pesticidalprotein. Signal peptides recognized by E. coli include the OmpA protein(Ghrayeb et al. (1984) EMBO J, 3: 2437-2442).

Pesticidal proteins and active variants thereof can be fermented in abacterial host and the resulting bacteria processed and used as amicrobial spray in the same manner that Bacillus thuringiensis strainshave been used as insecticidal sprays. In the case of a pesticidalprotein(s) that is secreted from Bacillus, the secretion signal isremoved or mutated using procedures known in the art. Such mutationsand/or deletions prevent secretion of the pesticidal protein(s) into thegrowth medium during the fermentation process. The pesticidal proteinsare retained within the cell, and the cells are then processed to yieldthe encapsulated pesticidal proteins.

Alternatively, the pesticidal proteins are produced by introducingheterologous genes into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated pesticidal proteins may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample U.S. Pat. No. 6,468,523 and U.S. Publication No. 20050138685,and the references cited therein. In the present invention, atransformed microorganism (which includes whole organisms, cells,spore(s), pesticidal protein(s), pesticidal component(s), pest-impactingcomponent(s), mutant(s), living or dead cells and cell components,including mixtures of living and dead cells and cell components, andincluding broken cells and cell components) or an isolated pesticidalprotein can be formulated with an acceptable carrier into a pesticidalor agricultural composition(s) that is, for example, a suspension, asolution, an emulsion, a dusting powder, a dispersible granule, awettable powder, and an emulsifiable concentrate, an aerosol, animpregnated granule, an adjuvant, a coatable paste, and alsoencapsulations in, for example, polymer substances.

Agricultural compositions may comprise a polypeptide, a recombinogenicpolypeptide or a variant or fragment thereof, as disclosed herein. Theagricultural composition disclosed herein may be applied to theenvironment of a plant or an area of cultivation, or applied to theplant, plant part, plant cell, or seed.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the present invention are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the present invention may beapplied to grain in preparation for or during storage in a grain bin orsilo, etc. The compositions of the present invention may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include, but are not limited to, foliar application,seed coating, and soil application. The number of applications and therate of application depend on the intensity of infestation by thecorresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate of dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate of primary composition thatrequires dilution with a suitable quantity of water or other diluantbefore application. The pesticidal concentration will vary dependingupon the nature of the particular formulation, specifically, whether itis a concentrate or to be used directly. The composition contains 1 to98% of a solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of asurfactant. These compositions will be administered at the labeled ratefor the commercial product, for example, about 0.01 lb-5.0 lb. per acrewhen in dry form and at about 0.01 pts.-10 pts. per acre when in liquidform.

In a further embodiment, the compositions, as well as the transformedmicroorganisms and pesticidal proteins, provided herein can be treatedprior to formulation to prolong the pesticidal activity when applied tothe environment of a target pest as long as the pretreatment is notdeleterious to the pesticidal activity. Such treatment can be bychemical and/or physical means as long as the treatment does notdeleteriously affect the properties of the composition(s). Examples ofchemical reagents include but are not limited to halogenating agents;aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, suchas zephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.).

In one aspect, pests may be killed or reduced in numbers in a given areaby application of the pesticidal proteins provided herein to the area.Alternatively, the pesticidal proteins may be prophylactically appliedto an environmental area to prevent infestation by a susceptible pest.Preferably the pest ingests, or is contacted with, apesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations or compositions may also vary with respect to climaticconditions, environmental considerations, and/or frequency ofapplication and/or severity of pest infestation.

The active ingredients are normally applied in the form of compositionsand can be applied to the crop area, plant, or seed to be treated.Methods are therefore provided for providing to a plant, plant cell,seed, plant part or an area of cultivation, an effective amount of theagricultural composition comprising the polypeptide, recombinogenicpolypeptide or an active variant or fragment thereof. By “effectiveamount” is intended an amount of a protein or composition has pesticidalactivity that is sufficient to kill or control the pest or result in anoticeable reduction in pest growth, feeding, or normal physiologicaldevelopment. Such decreases in numbers, pest growth, feeding or normaldevelopment can comprise any statistically significant decrease,including, for example a decrease of about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95% or greater.

For example, the compositions may be applied to grain in preparation foror during storage in a grain bin or silo, etc. The compositions may beapplied simultaneously or in succession with other compounds. Methods ofapplying an active ingredient or an agrochemical composition comprisingat least one of the polypeptides, recombinogenic polypeptides orvariants or fragments thereof as disclosed herein, include but are notlimited to, foliar application, seed coating, and soil application.

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with (or susceptible toinfestation by) a pest against which said polypeptide has pesticidalactivity. In some embodiments, the polypeptide has pesticidal activityagainst a lepidopteran, coleopteran, dipteran, hemipteran, or nematodepest, and said field is infested with a lepidopteran, hemipteran,coleopteran, dipteran, or nematode pest. As defined herein, the “yield”of the plant refers to the quality and/or quantity of biomass producedby the plant. By “biomass” is intended any measured plant product. Anincrease in biomass production is any improvement in the yield of themeasured plant product. Increasing plant yield has several commercialapplications. For example, increasing plant leaf biomass may increasethe yield of leafy vegetables for human or animal consumption.Additionally, increasing leaf biomass can be used to increase productionof plant-derived pharmaceutical or industrial products. An increase inyield can comprise any statistically significant increase including, butnot limited to, at least a 1% increase, at least a 3% increase, at leasta 5% increase, at least a 10% increase, at least a 20% increase, atleast a 30%, at least a 50%, at least a 70%, at least a 100% or agreater increase in yield compared to a plant not expressing thepesticidal sequence. In specific methods, plant yield is increased as aresult of improved pest resistance of a plant expressing a pesticidalprotein disclosed herein. Expression of the pesticidal protein resultsin a reduced ability of a pest to infest or feed.

The plants can also be treated with one or more chemical compositions,including one or more herbicide, insecticides, or fungicides.

Non-limiting embodiments include:

1. An isolated polypeptide having insecticidal activity, comprising

(a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of sequences set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108 109, 110, 111, 112,113, 114 115, 116, 117, 118, 119, 120 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, and/or 205; or

(b) a polypeptide comprising an amino acid sequence having at least thepercent sequence identity set forth in Table 1 to an amino acid sequenceselected from the group consisting of sequences set forth in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and/or 205.

2. The polypeptide of embodiment 1, wherein said polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, and/or 205.

3. A composition comprising the polypeptide of embodiments 1 or 2.

4. The polypeptide of embodiment 2, further comprising heterologousamino acid sequences.

5. A recombinant nucleic acid molecule that encodes the polypeptide ofembodiment 1, wherein said recombinant nucleic acid molecule is not thenaturally occurring sequence encoding said polypeptide.

6. The recombinant nucleic acid of embodiment 5, wherein said nucleicacid molecule is a synthetic sequence that has been designed forexpression in a plant.

7. The recombinant nucleic acid molecule of embodiment 6, wherein saidnucleic acid molecule is operably linked to a promoter capable ofdirecting expression in a plant cell.

8. The recombinant nucleic acid molecule of embodiment 5, wherein saidnucleic acid molecule is operably linked to a promoter capable ofdirecting expression in a bacteria.

9. A host cell that contains the recombinant nucleic acid molecule ofembodiment 8.

10. The host cell of embodiment 9, wherein said host cell is a bacterialhost cell.

11. A DNA construct comprising a promoter that drives expression in aplant cell operably linked to a recombinant nucleic acid moleculecomprising

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109 110, 111, 112 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205.

12. The DNA construct of embodiment 11, wherein said nucleotide sequenceis a synthetic DNA sequence that has been designed for expression in aplant.

13. A vector comprising the DNA construct of embodiment 11.

14. A host cell that contains the DNA construct of any one ofembodiments 11-13.

15. The host cell of embodiment 14, wherein the host cell is a plantcell.

16. A transgenic plant comprising the host cell of embodiment 15.

17. A composition comprising the host cell of embodiment 10.

18. The composition of embodiment 17, wherein said composition isselected from the group consisting of a powder, dust, pellet, granule,spray, emulsion, colloid, and solution.

19. The composition of embodiment 17, wherein said composition comprisesfrom about 1% to about 99% by weight of said polypeptide.

20. A method for controlling a pest population comprising contactingsaid population with a pesticidal-effective amount of the composition ofembodiment 17.

21. A method for killing a pest population comprising contacting saidpopulation with a pesticidal-effective amount of the composition ofembodiment 17.

22. A method for producing a polypeptide with pesticidal activity,comprising culturing the host cell of embodiment 9 under conditions inwhich the nucleic acid molecule encoding the polypeptide is expressed.

23. A plant having stably incorporated into its genome a DNA constructcomprising a nucleotide sequence that encodes a protein havingpesticidal activity, wherein said nucleotide sequence comprise

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, and/or 205; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205.

24. A transgenic seed of the plant of embodiment 23.

25. A method for protecting a plant from an insect pest, comprisingexpressing in a plant or cell thereof a nucleotide sequence that encodesa pesticidal polypeptide, wherein said nucleotide sequence comprising

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, and/or 205; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205.

26. The method of embodiment 25, wherein said plant produces apesticidal polypeptide having pesticidal against a lepidopteran orcoleopteran pest.

27. A method for increasing yield in a plant comprising growing in afield a plant or seed thereof having stably incorporated into its genomea DNA construct comprising a promoter that drives expression in a plantoperably linked to a nucleotide sequence that encodes a pesticidalpolypeptide, wherein said nucleotide sequence comprises

(a) a nucleotide sequence that encodes a polypeptide comprising theamino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, and/or 205; or,

(b) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least the percent sequence identity set forth inTable 1 to an amino acid sequence selected from the group consisting ofsequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205.

28. A method of obtaining a polynucleotide that encodes an improvedpolypeptide comprising pesticidal activity is provided, wherein theimproved polypeptide has at least one improved property over any one ofSEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, and/or 205comprising:

(a) recombining a plurality of parental polynucleotides comprising SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134,135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,and/or 205 or an active variant or fragment thereof to produce a libraryof recombinant polynucleotides encoding recombinant pesticidalpolypeptides;

(b) screening the library to identify a recombinant polynucleotide thatencodes an improved recombinant pesticidal polypeptide that has anenhanced property improved over the parental polynucleotide;

(c) recovering the recombinant polynucleotide that encodes the improvedrecombinant pesticidal polypeptide identified in (b); and,

(d) repeating steps (a), (b) and (c) using the recombinantpolynucleotide recovered in step (c) as one of the plurality of parentalpolynucleotides in repeated step (a).

The following examples are offered by way of illustration and not by wayof limitation.

Experimental EXAMPLE 1 Discovery of Novel Genes by Sequencing and DNAAnalysis

Microbial cultures were grown in liquid culture in standard laboratorymedia. Cultures were grown to saturation (16 to 24 hours) before DNApreparation. DNA was extracted from bacterial cells by detergent lysis,followed by binding to a silica matrix and washing with an ethanolbuffer. Purified DNA was eluted from the silica matrix with a mildlyalkaline aqueous buffer.

DNA for sequencing was tested for purity and concentration byspectrophotometry. Sequencing libraries were prepared using the NexteraXT library preparation kit according to the manufacturer's protocol.Sequence data was generated on a HiSeq 2000 according to the IlluminaHiSeq 2000 System User Guide protocol.

Sequencing reads were assembled into draft genomes using the CLC BioAssembly Cell software package. Following assembly, gene calls were madeby several methods and resulting gene sequences were interrogated toidentify novel homologs of pesticidal genes. Novel genes were identifiedby BLAST, by domain composition, and by pairwise alignment versus atarget set of pesticidal genes. A summary of such sequences is set forthin Table 1 and 3.

Genes identified in the homology search were amplified from bacterialDNA by PCR and cloned into bacterial expression vectors containing fusedin-frame purification tags. Cloned genes were expressed in E. coli andpurified by column chromatography. Purified proteins were assessed ininsect diet bioassay studies to identify active proteins.

EXAMPLE 2 Heterologous Expression in E. coli

Each open reading frame set forth in SEQ ID NO: 1-205 is cloned into anE. coli expression vector containing a maltose binding protein (pMBP).The expression vector is transformed into BL21*RIPL. An LB culturesupplemented with carbenicillin is inoculated with a single colony andgrown overnight at 37 degrees C. using 0.5% of the overnight culture, afresh culture is inoculated and grown to logarithmic phase at 37 degreesC. The culture is induced using 250 mM IPTG for 18 hours at 16 degreesC. The cells are pelleted and resuspended in 10 mM Tris pH7.4 and 150 mMNaCl supplemented with protease inhibitors. The protein expression isevaluated by SDS-PAGE.

EXAMPLE 3 Pesticidal Activity Against Coleopteran and Lepidoptera

Methods

Protein Expression: Each sequence set forth in SEQ ID NO: 1-205 isexpressed in E. coli as described in Example 2. 400 mL of LB isinoculated and grown to an OD600 of 0.6. The culture is induced with0.25 mM IPTG overnight at 16 C. The cells are spun down and the cellpellet is resuspend in 5 mL of buffer. The resuspension is sonicated for2 min on ice.

Bioassay: Fall army worm (FAW), corn ear worm (CEW), European corn borer(ECB) southwestern corn borer (SWCB) and diamond backed moth (DBM) eggsare purchased from a commercial insectary (Benzon Research Inc.,Carlisle, Pa.). The FAW, CEW, ECB and BCW eggs are incubated to thepoint that eclosion would occur within 12 hrs of the assay setup. SWCBand DBM are introduced to the assay as neonate larvae. Assays arecarried out in 24-well trays containing multispecies lepidopteran diet(SOUTHLAND PRODUCTS INCORPORATED, Lake Village, Ark.). Samples of thesonicated lysate are applied to the surface of the diet (diet overlay)and allowed to evaporate and soak into the diet. For CEW, FAW, BCW, ECBand SWCB, a 125 μl of sonicated lysate is added to the diet surface anddried. For DBM, 50 μl of a 1:2 dilution of sonicated lysate was added tothe diet surface. The bioassay plates are sealed with a plate sealingfilm vented with pin holes. The plates are incubated at 26 C at 65% RHon a 16:8 day:night cycle in a Percival for 5 days. The assays areassessed for level of mortality, growth inhibition and feedinginhibition.

For the western corn rootworm bioassay, the protein construct/lysate isevaluated in an insect bioassay by dispensing 60 μl volume on the topsurface of diet in well/s of 24-well plate (Cellstar, 24-well, GreinerBio One) and allowed to dry. Each well contains 500 μl diet (Marrone etal., 1985). Fifteen to twenty neonate larvae are introduced in each wellusing a fine tip paint brush and the plate is covered with membrane(Viewseal, Greiner Bio One). The bioassay is stored at ambienttemperature and scored for mortality, and/or growth/feeding inhibitionat day 4.

For Colorado Potato Beetle (CPB) a cork bore size No. 8 leaf disk isexcised from potato leaf and is dipped in the protein construct/lysateuntil thoroughly wet and placed on top of filter disk (Millipore, glassfiber filter, 13 mm). Sixty μl dH₂O is added to each filter disk andplaced in each well of 24-well plate (Cellstar, 24-well, Greiner BioOne). The leaf disk is allowed to dry and five to seven first instarlarvae are introduced in each well using a fine tip paint brush. Theplate is covered with membrane (Viewseal, Greiner Bio One) and smallhole is punctured in each well of the membrane. The construct isevaluated with four replicates, and scored for mortality and leaf damageon day 3.

EXAMPLE 4 Pesticidal Activity Against Hemipteran

Protein Expression: Each of the sequences set forth in SEQ ID NO: 1-205is expressed in E. coli as described in Example 2. 400 mL of LB isinoculated and grown to an OD600 of 0.6. The culture is induced with0.25 mM IPTG overnight at 16 C. The cells are spun down and the cellpellet is re-suspend in 5 mL of buffer. The resuspension is sonicatedfor 2 min on ice.

Second instar SGSB are obtained from a commercial insectary (BenzonResearch Inc., Carlisle, Pa.). A 50% v/v ratio of sonicated lysatesample to 20% sucrose is employed in the bioassay. Stretched parafilm isused as a feeding membrane to expose the SGSB to the diet/samplemixture. The plates are incubated at 25 C:21 C, 16:8 day:night cycle at65% RH for 5 days.

Mortality is scored for each sample.

EXAMPLE 5 Transformation of Soybean

DNA constructs comprising each of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 189, 189, 190, 191, 192, and/or 193 or activevariants or fragments thereof operably linked to a promoter active in aplant are cloned into transformation vectors and introduced intoAgrobacterium as described in U.S. Provisional Application No.62/094,782, filed Dec. 19, 2015, herein incorporated by reference in itsentirety.

Four days prior to inoculation, several loops of Agrobacterium arestreaked to a fresh plate of YEP* medium supplemented with theappropriate antibiotics** (spectinomycin, chloramphenicol andkanamycin). Bacteria are grown for two days in the dark at 28 C. Aftertwo days, several loops of bacteria are transferred to 3 ml of YEPliquid medium with antibiotics in a 125 ml Erlenmeyer flask. Flasks areplaced on a rotary shaker at 250 RPM at 28 C overnight. One day beforeinoculation, 2-3 ml of the overnight culture were transferred to 125 mlof YEP with antibiotics in a 500 ml Erlenmeyer flask. Flasks are placedon a rotary shaker at 250 RPM at 28 C overnight.

Prior to inoculation, the OD of the bacterial culture is checked at OD620. An OD of 0.8-1.0 indicates that the culture is in log phase. Theculture is centrifuged at 4000 RPM for 10 minutes in Oakridge tubes. Thesupernatant is discarded and the pellet is re-suspended in a volume ofSoybean Infection Medium (SI) to achieve the desired OD. The culturesare held with periodic mixing until needed for inoculation.

Two or three days prior to inoculation, soybean seeds are surfacesterilized using chlorine gas. In a fume hood, a petri dish with seedsis place in a bell jar with the lid off. 1.75 ml of 12 N HCl is slowlyadded to 100 ml of bleach in a 250 ml Erlenmeyer flask inside the belljar. The lid is immediately placed on top of the bell jar. Seeds areallowed to sterilize for 14-16 hours (overnight). The top is removedfrom the bell jar and the lid of the petri dish is replaced. The petridish with the surface sterilized is then opened in a laminar flow foraround 30 minutes to disperse any remaining chlorine gas.

Seeds are imbibed with either sterile DI water or soybean infectionmedium (SI) for 1-2 days. Twenty to 30 seeds are covered with liquid ina 100×25 mm petri dish and incubated in the dark at 24 C. Afterimbibition, non-germinating seeds are discarded.

Cotyledonary explants is processed on a sterile paper plate with sterilefilter paper dampened using SI medium employing the methods of U.S. Pat.No. 7,473,822, herein incorporated by reference.

Typically, 16-20 cotyledons are inoculated per treatment. The SI mediumused for holding the explants is discarded and replaced with 25 ml ofAgrobacterium culture (OD 620=0.8−20). After all explants are submerged,the inoculation is carried out for 30 minutes with periodic swirling ofthe dish. After 30 minutes, the Agrobacterium culture is removed.

Co-cultivation plates is prepared by overlaying one piece of sterilepaper onto Soybean Co-cultivation Medium (SCC). Without blotting, theinoculated cotyledons is cultured adaxial side down on the filter paper.Around 20 explants can be cultured on each plate. The plates are sealedwith Parafilm and cultured at 24 C and around 120 umoles m-2s-1 (in aPercival incubator) for 4-5 days.

After co-cultivation, the cotyledons are washed 3 times in 25 ml ofSoybean Wash Medium with 200 mg/l of cefotaxime and timentin. Thecotyledons are blotted on sterile filter paper and then transferred toSoybean Shoot Induction Medium (SSI). The nodal end of the explant isdepressed slightly into the medium with distal end kept above thesurface at about 45 deg. No more than 10 explants are cultured on eachplate. The plates are wrapped with Micropore tape and cultured in thePercival at 24 C and around 120 umoles m-2s-1.

The explants are transferred to fresh SSI medium after 14 days. Emergingshoots from the shoot apex and cotyledonary node are discarded. Shootinduction is continued for another 14 days under the same conditions.

After 4 weeks of shoot induction, the cotyledon is separated from thenodal end and a parallel cut is made underneath the area of shootinduction (shoot pad). The area of the parallel cut is placed on SoybeanShoot Elongation Medium (SSE) and the explants cultured in the Percivalat 24 C and around 120 umoles m-2s-1. This step is repeated every twoweeks for up to 8 weeks as long as shoots continue to elongate.

When shoots reach a length of 2-3 cm, they are transferred to SoybeanRooting Medium (SR) in a Plantcon vessel and incubated under the sameconditions for 2 weeks or until roots reach a length of around 3-4 cm.After this, plants are transferred to soil.

Note, all media mentioned for soybean transformation are found in Paz etal. (2010) Agrobacterium-mediated transformation of soybean and recoveryof transgenic soybean plants; Plant Transformation Facility of IowaState University, which is herein incorporated by reference in itsentirety. (See, agron-www.agron.iastate.edu/ptf/protocol/Soybean.pdf.)

EXAMPLE 6 Transformation of Maize

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000.times. Stock) N6 Vitamins; 800 mg/LL-Asparagine; 100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/LCasamino acids; 50 g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D).However, media and salts other than DN62A5 S are suitable and are knownin the art. Embryos are incubated overnight at 25 degree C. in the dark.However, it is not necessary per se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842). DNA constructs designed toexpress the GRG proteins of the present invention in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for about 30 min on osmotic media,and placed onto incubation media overnight at 25 degree C. in the dark.To avoid unduly damaging beamed explants, they are incubated for atleast 24 hours prior to transfer to recovery media. Embryos are thenspread onto recovery period media, for about 5 days, 25 degree C. in thedark, then transferred to a selection media. Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated by methods known inthe art. The resulting shoots are allowed to root on rooting media, andthe resulting plants are transferred to nursery pots and propagated astransgenic plants.

EXAMPLE 7 Pesticidal Activity Against Nematodes Heterodera Glycine's(Soybean Cyst Nematode) In-Vitro Assay.

Soybean Cyst Nematodes are dispensed into a 96 well assay plate with atotal volume of 100 uls and 100 J2 per well. The protein of interest asset forth in any one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, and/or 205 is dispensed into the wells andheld at room temperature for assessment. Finally the 96 well platecontaining the SCN J2 is analyzed for motility. Data is reported as %inhibition as compared to the controls. Hits are defined as greater orequal to 70% inhibition.

Heterodera Glycine's (Soybean Cyst Nematode) On-Plant Assay

Soybean plants expressing one or more of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, and/or 205 are generated asdescribed elsewhere herein. A 3-week-old soybean cutting is inoculatedwith 5000 SCN eggs per plant. This infection is held for 70days and thenharvested for counting of SCN cyst that has developed on the plant. Datais reported as % inhibition as compared to the controls. Hits aredefined as greater or equal to 90% inhibition.

Meloidogyne Incognita (Root-Knot Nematode) In-Vitro Assay

Root-Knot Nematodes are dispensed into a 96 well assay plate with atotal volume of 100 uls and 100 J2 per well. The protein of interestcomprising any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, and/or 205 is dispensed into the wells and heldat room temperature for assessment. Finally the 96 well plate containingthe RKN J2 is analyzed for motility. Data is reported as % inhibition ascompared to the controls. Hits are defined as greater or equal to 70%inhibition.

Meloidogyne Incognita (Root-Knot Nematode) On-Plant Assay

Soybean plants expressing one or more of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, and/or 205 are generated asdescribed elsewhere herein. A 3-week-old soybean is inoculated with 5000RKN eggs per plant. This infection is held for 70days and then harvestedfor counting of RKN eggs that have developed in the plant. Data isreported as % inhibition as compared to the controls. Hits are definedas greater or equal to 90% inhibition.

EXAMPLE 8 Additional Assays for Pesticidal Activity

The various polypeptides set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 189, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, and/or 205 can be tested to actas a pesticide upon a pest in a number of ways. One such method is toperform a feeding assay. In such a feeding assay, one exposes the pestto a sample containing either compounds to be tested or control samples.Often this is performed by placing the material to be tested, or asuitable dilution of such material, onto a material that the pest willingest, such as an artificial diet. The material to be tested may becomposed of a liquid, solid, or slurry. The material to be tested may beplaced upon the surface and then allowed to dry. Alternatively, thematerial to be tested may be mixed with a molten artificial diet, andthen dispensed into the assay chamber. The assay chamber may be, forexample, a cup, a dish, or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson and Preisler, eds. (1992)Pesticide bioassays with arthropods, CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals ArthropodManagement Tests and Journal of Economic Entomology or by discussionwith members of the Entomological Society of America (ESA). Any one ofSEQ ID NOS: 1-205 can be expressed and employed in an assay as set forthin Examples 3 and 4, herein.

EXAMPLE 9 Pesticidal Activity Against Coleopteran and Lepidoptera

Protein Expression: Each sequence set forth in Table 3 was expressed inE. coli as described in Example 2. 400 mL of LB was inoculated and grownto an OD600 of 0.6. The culture was induced with 0.25 mM IPTG overnightat 16° C. The cells were spun down and the cell pellet was resuspendedin 5 mL of buffer. The resuspension was sonicated for 2 min on ice.

Bioassay: Fall army worm (FAW), corn ear worm (CEW), European corn borer(ECB) southwestern corn borer (SWCB) and diamond backed moth (DBM or Px)eggs were purchased from a commercial insectary (Benzon Research Inc.,Carlisle, Pa.). The FAW, CEW, ECB and BCW eggs were incubated to thepoint that eclosion would occur within 12 hrs of the assay setup. SWCBand DBM were introduced to the assay as neonate larvae. Assays werecarried out in 24-well trays containing multispecies lepidopteran diet(Southland Products Inc., Lake Village, Ark.). Samples of the sonicatedlysate were applied to the surface of the diet (diet overlay) andallowed to evaporate and soak into the diet. For CEW, FAW, BCW, ECB andSWCB, a 125 μl of sonicated lysate was added to the diet surface anddried. For DBM, 50 μl of a 1:2 dilution of sonicated lysate was added tothe diet surface. The bioassay plates were sealed with a plate sealingfilm vented with pin holes. The plates were incubated at 26° C. at 65%relative humidity (RH) on a 16:8 day:night cycle in a Percival for 5days. The assays were assessed for level of mortality, growth inhibitionand feeding inhibition.

For the western corn rootworm (WCR) bioassay, the proteinconstruct/lysate was evaluated in an insect bioassay by dispensing 60 μlvolume on the top surface of diet in well/s of 24-well plate (Cellstar,24-well, Greiner Bio One) and allowed to dry. Each well contained 500 μldiet (Marrone et al., 1985). Fifteen to twenty neonate larvae wereintroduced in each well using a fine tip paint brush and the plate wascovered with membrane (Viewseal, Greiner Bio One). The bioassay wasstored at ambient temperature and scored for mortality, and/orgrowth/feeding inhibition at day 4.

For Colorado Potato Beetle (CPB) a cork bore size No. 8 leaf disk wasexcised from potato leaf and was dipped in the protein construct/lysateuntil thoroughly wet and is placed on top of filter disk (Millipore,glass fiber filter, 13 mm). 60 μl dH₂O was added to each filter disk andplaced in each well of 24-well plate (Cellstar, 24-well, Greiner BioOne). The leaf disk was allowed to dry and five to seven first instarlarvae were introduced in each well using a fine tip paint brush. Theplate was covered with a membrane (Viewseal, Greiner Bio One) and asmall hole was punctured in each well of the membrane. The construct wasevaluated with four replicates, and scored for mortality and leaf damageon day 3.

Table 3 provides a summary of pesticidal activity against coleopteranand lepidoptera of the various sequences. Table code: “−” indicates noactivity seen; “+” indicates pesticidal activity; “NT” indicates nottested; “S” indicates stunt; “SS” indicates slight stunt; “HM” indicateshigh mortality; “LF” indicates low feeding; “M” indicates mortality.

TABLE 3 Summary of Pesticidal Activity against Coleopteran andLepidoptera. APG Seq ID FAW CEW BCW ECB SWCB CPB Px WCRW APG09492.1 SeqID 159 − NT NT NT NT NT NT − APG02013.1 Seq ID 21  M, S NT NT NT NT NTNT − APG09953.1 Seq ID 168 − NT NT NT NT NT NT − APG09211.1 Seq ID 148 −NT NT NT NT NT NT − APG06228.1 Seq ID 88  SS NT NT NT NT NT NT −APG03324.1 Seq ID 55  SS NT NT NT NT NT NT − APG05341.1 Seq ID 78  − NTNT NT NT NT NT − APG01189.1 Seq ID 7  M, S NT NT NT NT NT NT −APG03900.0 Seq ID 60  SS NT NT NT NT NT NT − APG07889.1 Seq ID 127 − NTNT NT NT NT NT − APG07679.1 Seq ID 122 − NT NT NT NT NT NT − APG01013.1Seq ID 3  − NT NT NT NT NT NT − APG07383.1 Seq ID 113 SS NT NT NT NT NTNT − APG02438.1 Seq ID 30  − NT NT NT NT NT NT − APG06730.1 Seq ID 97 M, S NT NT NT NT NT NT − APG02742.1 Seq ID 41  − NT NT NT NT NT NT −APG04586.0 Seq ID 66  SS NT NT NT NT NT NT − APG02701.1 Seq ID 39  − NTNT NT NT NT NT − APG01844.1 Seq ID 13  − NT NT NT NT NT NT − APG09587.0Seq ID 14  M, S NT NT NT NT NT NT − APG07491.1 Seq ID 115 SS NT NT NT NTNT NT − APG08361.1 Seq ID 134 − NT NT NT NT NT NT − APG06801.1 Seq ID99  − NT NT NT NT NT NT − APG05259.1 Seq ID 73  − NT NT NT NT NT NT −APG01363.1 Seq ID 9  − NT NT NT NT NT NT − APG03187.1 Seq ID 49  SS NTNT NT NT NT NT − APG02403.1 Seq ID 27  − NT NT NT NT NT NT − APG07980.0Seq ID 28  − NT NT NT NT NT NT − APG04671.1 Seq ID 68  − NT NT NT NT NTNT − APG03815.0 Seq ID 69  − NT NT NT NT NT NT − APG04415.0 Seq ID 65 SS NT NT NT NT NT NT − APG09604.1 Seq ID 161 − NT NT NT NT NT NT −APG05886.1 Seq ID 81  − NT NT NT NT NT NT − APG03498.1 Seq ID 57  − NTNT NT NT NT NT − APG05311.1 Seq ID 76  − NT NT NT NT NT NT − APG09977.1Seq ID 170 M, S NT NT NT NT NT NT − APG08005.1 Seq ID 129 − NT NT NT NTNT NT − APG06401.1 Seq ID 93  − NT NT NT NT NT NT − APG02647.1 Seq ID37  M, S NT NT NT NT NT NT − APG07125.1 Seq ID 105 − NT NT NT NT NT NT −APG08369.1 Seq ID 136 M, S NT NT NT NT NT NT − APG03101.1 Seq ID 46  M,S NT NT NT NT NT NT − APG04345.1 Seq ID 64  SS NT NT NT NT NT NT −APG08835.1 Seq ID 140 − NT NT NT NT NT NT − APG07295.0 Seq ID 124 − NTNT NT NT NT NT − APG07799.0 Seq ID 123 − NT NT NT NT NT NT − APG08452.0Seq ID 138 M, S NT NT NT NT NT NT − APG09155.1 Seq ID 146 − NT NT NT NTNT NT − APG09869.0 Seq ID 164 − NT NT NT NT NT NT − APG09869.1 Seq ID165 − NT NT NT NT NT NT − APG09869.2 Seq ID 166 − NT NT NT NT NT NT −APG02060.1 Seq ID 23  − NT NT NT NT NT NT − APG09459.0 Seq ID 153 M, SNT NT NT NT NT NT − APG09459.1 Seq ID 154 M, S NT NT NT NT NT NT −APG09459.2 Seq ID 155 M, S NT NT NT NT NT NT − APG06560.1 Seq ID 95  SNT NT NT NT NT NT − APG07247.1 Seq ID 109 − NT NT NT NT NT NT −APG09768.1 Seq ID 163 − NT NT NT NT NT NT − APG02572.1 Seq ID 32  − NTNT NT NT NT NT − APG03147.0 Seq ID 47  − NT NT NT NT NT NT − APG05669.0Seq ID 79  M, S NT NT NT NT NT NT − APG08992.1 Seq ID 142 M, S NT NT NTNT NT NT − APG04069.1 Seq ID 62  SS NT NT NT NT NT NT − APG07235.1 SeqID 107 M, S NT NT NT NT NT NT − APG07083.0 Seq ID 103 − NT NT NT NT NTNT − APG03889.1 Seq ID 59  − NT NT NT NT NT NT − APG05896.0 Seq ID 82  −NT NT NT NT NT NT − APG03279.0 Seq ID 53  − NT NT NT NT NT NT −APG05284.0 Seq ID 74  SS NT NT NT NT NT NT − APG09338.1 Seq ID 150 M, SNT NT NT NT NT NT − APG06242.1 Seq ID 90  − NT NT NT NT NT NT −APG06242.2 Seq ID 91  − NT NT NT NT NT NT − APG09100.1 Seq ID 144 SS NTNT NT NT NT NT − APG07606.1 Seq ID 119 — NT NT NT NT NT NT − APG07606.2Seq ID 120 M, S NT NT NT NT NT NT − APG09460.1 Seq ID 157 − NT NT NT NTNT NT − APG03055.1 Seq ID 43  − NT NT NT NT NT NT − APG03055.2 Seq ID44  − NT NT NT NT NT NT − APG01969.1 Seq ID 17  − NT NT NT NT NT NT −APG01971.1 Seq ID 19  S − − NT NT − NT − APG01502.1 Seq ID 11  SS − − NTNT − NT − APG06971.1 Seq ID 102 M, S NT NT NT NT NT NT − APG02630.1 SeqID 34  SS NT NT NT NT NT NT − APG02630.2 Seq ID 35  SS NT NT NT NT NT NT− APG09441.1 Seq ID 152 − NT NT NT NT NT NT − APG08430.0 Seq ID 137 − NTNT NT NT NT NT − APG04699.0 Seq ID 70  M, S NT NT NT NT NT NT −APG02123.1 Seq ID 25  M, S NT NT NT NT NT NT − APG01155.1 Seq ID 5  SSNT NT NT NT NT NT − APG00837.0 Seq ID 1  HM, S NT NT NT NT NT NT −APG07334.1 Seq ID 111 SS NT NT NT NT NT NT − APG09329.2 Seq ID 193 − NTNT NT NT NT NT − APG07868.0 Seq ID 125 − NT NT NT NT NT NT − APG08195.0Seq ID 130 − NT NT NT NT NT NT − APG06043.1 Seq ID 84  − NT NT NT NT NTNT − APG04912.0 Seq ID 71  − NT NT NT NT NT NT − APG01927.0 Seq ID 15  −NT NT NT NT NT NT − APG07526.1 Seq ID 117 − NT NT NT NT NT NT −APG03195.1 Seq ID 51  − NT NT NT NT NT NT − APG03195.2 Seq ID 52  − NTNT NT NT NT NT − APG06963.0 Seq ID 100 − NT NT NT NT NT NT − APG08330.1Seq ID 132 − NT NT NT NT NT NT − APG06187.1 Seq ID 86  − NT NT NT NT NTNT − APG05956.1 Seq ID 185 − NT NT NT NT NT NT − APG06419.1 Seq ID 187SS NT NT NT NT NT NT − APG05791.1 Seq ID 183 SS NT NT NT NT NT NT −APG01488.0 Seq ID 177 SS NT NT NT NT NT NT − APG06727.2 Seq ID 190 S NTNT NT NT NT NT − APG00765.2 Seq ID 173 SS NT NT NT NT NT NT − APG04947.1Seq ID 181 SS SS − NT NT − NT + APG01166.2 Seq ID 176 S NT NT NT NT NTNT − APG03067.1 Seq ID 179 S NT NT NT NT NT NT − APG06237.0 Seq ID 197SS NT NT NT NT NT NT − APG03898.2 Seq ID 196 − NT NT NT NT NT NT −

EXAMPLE 10 Pesticidal Activity Against Hemipteran

Protein Expression: Each of the sequences set forth in Table 4 wasexpressed in E. coli as described in Example 2. 400 mL of LB wasinoculated and grown to an OD600 of 0.6. The culture was induced with0.25 mM IPTG overnight at 16° C. The cells were spun down and the cellpellet was re-suspended in 5 mL of buffer. The resuspension wassonicated for 2 min on ice.

Second instar southern green stinkbug (SGSB) were obtained from acommercial insectary (Benzon Research Inc., Carlisle, Pa.). A 50% v/vratio of sonicated lysate sample to 20% sucrose was employed in thebioassay. Stretched parafilm was used as a feeding membrane to exposethe SGSB to the diet/sample mixture. The plates were incubated at 25°C.: 21° C., 16:8 day:night cycle at 65% RH for 5 days.

Mortality was scored for each sample. The results are set forth in Table4. A dashed line indicates no mortality was detected. The proteinslisted in Table 4 showed from about 10% to about 100% mortality, 25%mortality, or 50% mortality (2 stinkbug out of 4 died) (as indicated)against southern green stinkbug. The negative controls (empty vectorexpressed binding domain and buffer only) both showed no mortality (0stinkbugs out of 4).

TABLE 4 Summary of Pesticidal Activity against Hemipteran APG Seq IDSGSB APG09492.1 Seq ID 159 30 APG02013.1 Seq ID 21 10 APG09953.1 Seq ID168 20 APG09211.1 Seq ID 148 50 APG06228.1 Seq ID 88 30 APG03324.1 SeqID 55 30 APG05341.1 Seq ID 78 20 APG01189.1 Seq ID 7 10 APG03900.0 SeqID 60 0 APG07889.1 Seq ID 127 70 APG07679.1 Seq ID 122 50 APG01013.1 SeqID 3 50 APG07383.1 Seq ID 113 30 APG02438.1 Seq ID 30 30 APG06730.1 SeqID 97 40 APG02742.1 Seq ID 41 50 APG04586.0 Seq ID 66 50 APG02701.1 SeqID 39 0 APG01844.1 Seq ID 13 30 APG09587.0 Seq ID 14 10 APG07491.1 SeqID 115 10 APG08361.1 Seq ID 134 30 APG06801.1 Seq ID 99 30 APG05259.1Seq ID 73 20 APG01363.1 Seq ID 9 50 APG03187.1 Seq ID 49 30 APG02403.1Seq ID 27 0 APG07980.0 Seq ID 28 10 APG04671.1 Seq ID 68 10 APG03815.0Seq ID 69 30 APG04415.0 Seq ID 65 50 APG09604.1 Seq ID 161 30 APG05886.1Seq ID 81 50 APG03498.1 Seq ID 57 30 APG05311.1 Seq ID 76 50 APG09977.1Seq ID 170 50 APG08005.1 Seq ID 129 60 APG06401.1 Seq ID 93 40APG02647.1 Seq ID 37 50 APG07125.1 Seq ID 105 0 APG08369.1 Seq ID 136 30APG03101.1 Seq ID 46 0 APG04345.1 Seq ID 64 20 APG08835.1 Seq ID 140 20APG07295.0 Seq ID 124 30 APG07799.0 Seq ID 123 30 APG08452.0 Seq ID 13850 APG09155.1 Seq ID 146 50 APG09869.0 Seq ID 164 30 APG09869.1 Seq ID165 20 APG09869.2 Seq ID 166 40 APG02060.1 Seq ID 23 10 APG09459.0 SeqID 153 40 APG09459.1 Seq ID 154 20 APG09459.2 Seq ID 155 40 APG06560.1Seq ID 95 0 APG07247.1 Seq ID 109 50 APG09768.1 Seq ID 163 30 APG02572.1Seq ID 32 60 APG03147.0 Seq ID 47 20 APG05669.0 Seq ID 79 60 APG08992.1Seq ID 142 30 APG04069.1 Seq ID 62 30 APG07235.1 Seq ID 107 0 APG07083.0Seq ID 103 30 APG03889.1 Seq ID 59 30 APG05896.0 Seq ID 82 80 APG03279.0Seq ID 53 20 APG05284.0 Seq ID 74 30 APG09338.1 Seq ID 150 30 APG06242.1Seq ID 90 60 APG06242.2 Seq ID 91 40 APG09100.1 Seq ID 144 40 APG07606.1Seq ID 119 20 APG07606.2 Seq ID 120 50 APG09460.1 Seq ID 157 40APG03055.1 Seq ID 43 40 APG03055.2 Seq ID 44 40 APG01969.1 Seq ID 17 50APG01971.1 Seq ID 19 20 APG01502.1 Seq ID 11 20 APG06971.1 Seq ID 102 50APG02630.1 Seq ID 34 20 APG02630.2 Seq ID 35 40 APG09441.1 Seq ID 152 30APG08430.0 Seq ID 137 20 APG04699.0 Seq ID 70 30 APG02123.1 Seq ID 25 40APG01155.1 Seq ID 5 50 APG00837.0 Seq ID 1 50 APG07334.1 Seq ID 111 40APG09329.2 Seq ID 193 40 APG07868.0 Seq ID 125 40 APG08195.0 Seq ID 13060 APG06043.1 Seq ID 84 50 APG04912.0 Seq ID 71 40 APG01927.0 Seq ID 1540 APG07526.1 Seq ID 117 50 APG03195.1 Seq ID 51 50 APG03195.2 Seq ID 5230 APG06963.0 Seq ID 100 30 APG08330.1 Seq ID 132 20 APG06187.1 Seq ID86 30 APG05956.1 Seq ID 185 0 APG06419.1 Seq ID 187 20 APG05791.1 Seq ID183 0 APG01488.0 Seq ID 177 0 APG06727.2 Seq ID 190 0 APG00765.2 Seq ID173 10 APG04947.1 Seq ID 181 100 APG01166.2 Seq ID 176 40 APG03067.1 SeqID 179 0 APG06237.0 Seq ID 197 NT APG03898.2 Seq ID 196 NT

EXAMPLE 11 Pesticidal Activity Against Soybean Aphid

Protein Expression: Each sequence set forth in SEQ ID NOs: 1, 3, 5, 7,9, 11, 13, 14, 15, 17, 19, 21, 23, 25, 27, 28, 30, 32, 34, 35, 37, 39,41, 43, 44, 46, 47, 49, 51, 52, 53, 55, 57, 59, 60, 62, 64, 65, 66, 68,69, 70, 71, 73, 74, 76, 78, 79, 81, 82, 84, 86, 88, 90, 91, 93, 95, 97,99, 100, 102, 103, 105, 107, 109, 111, 113, 115, 117, 119, 120, 122,123, 124, 125, 127, 129, 130, 132, 134, 136, 137, 138, 140, 142, 144,146, 148, 150, 152, 153, 154, 155, 157, 159, 161, 163, 164, 165, 166,168, 170, 173, 176, 177, 179, 181, 183, 185, 187, 190, and 193 (or anactive variant or fragment thereof) was expressed in E. coli asdescribed in Example 2. 400 mL of LB was inoculated and grown to anOD600 of 0.6. The culture was induced with 0.25 mM IPTG overnight at 16°C. The cells were spun down and the cell pellet was resuspended in 5 mLof buffer. The resuspension was sonicated for 2 min on ice.

Soybean aphids (SBA) were obtained from Michigan State University. Sixadult aphids were added to each well of a 24 well plate. Purifiedproteins were provided in liquid artificial diet at a rate of 25% (50 ulprotein, 150 ul artificial diet), and were sealed with an artificialmembrane through which the aphids are able to feed. The plates were heldin an incubator at 26° C., 60% RH, 16:8 day:night cycle for 5 days.Mortality, feeding, and reproduction were scored for each sample on days3, 4, and 5 post-treatment. Mortality was calculated as percent dead ofthe original 6 adult aphids.

Feed and reproduction activities were assigned a score on a 0-3 pointscale with 0 being no feeding or reproduction and 3 being high feedingor reproduction. Feeding was measured as the amount of honeydew (liquidexcretions produced by aphids) that collected in each well. Reproductionwas the number of live immature aphids that were observed in each well.Mortality, feeding, and reproduction data were assessed using a combinedscoring system to establish cutoff levels for activity. The combinedscore was calculated as:

Combined Score=(Feeding+Reproduction+Mortality Score)/3, where MortalityScore=3−(3 x % Mortality/100). The results are set forth in Table 6. “+”indicates pesticidal activity.

TABLE 5 Cut-offs used to rate individual wells as active or not for eachobservational day post-introduction of aphids to assay wells. A well isdeemed active if measure ≤ cut-off. Cut-off used to designate activityActivity Measure Day 3 Day 4 Day 5 Combined Score 2 1.5 1

TABLE 6 Summary of Pesticidal Activity against soybean aphid APG SEQ IDTested against SBA APG04947.1 SEQ ID 181 +

SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14, 15, 17, 19, 21, 23, 25, 27, 28,30, 32, 34, 35, 37, 39, 41, 43, 44, 46, 47, 49, 51, 52, 53, 55, 57, 59,60, 62, 64, 65, 66, 68, 69, 70, 71, 73, 74, 76, 78, 79, 81, 82, 84, 86,88, 90, 91, 93, 95, 97, 99, 100, 102, 103, 105, 107, 109, 111, 113, 115,117, 119, 120, 122, 123, 124, 125, 127, 129, 130, 132, 134, 136, 137,138, 140, 142, 144, 146, 148, 150, 152, 153, 154, 155, 157, 159, 161,163, 164, 165, 166, 168, 170, 173, 176, 177, 179, 183, 185, 187, 190,and 193 were tested and did not have activity in this experiment.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed is:
 1. A recombinant polypeptide having pesticidal activity, said polypeptide comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and/or 205, wherein said polypeptide has pesticidal activity, and wherein said polypeptide further comprises a heterologous amino acid sequence chemically linked to said polypeptide.
 2. The polypeptide of claim 1, further comprising a heterologous amino acid sequence.
 3. A composition comprising the polypeptide of claim
 1. 4. A recombinant nucleic acid molecule encoding a polypeptide comprising: an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, or 205; wherein said polypeptide has pesticidal activity; wherein said recombinant nucleic acid molecule is operably linked to a heterologous promoter.
 5. The recombinant nucleic acid molecule of claim 4, wherein said recombinant nucleic acid molecule is a synthetic sequence designed for expression in a plant.
 6. The recombinant nucleic acid molecule of claim 4, wherein said heterologous promoter is capable of directing expression in a plant cell.
 7. The recombinant nucleic acid molecule of claim 4, wherein said heterologous promoter is capable of directing expression in a bacterium.
 8. A host cell comprising the recombinant nucleic acid molecule of claim
 4. 9. The host cell of claim 8, wherein said host cell is a bacterial host cell.
 10. A DNA construct comprising a promoter that drives expression in a plant cell operably linked to a recombinant nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to an amino acid sequence set forth in SEQ ID Nos: selected from the group consisting of sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, wherein said polypeptide has pesticidal activity.
 11. The DNA construct of claim 10, wherein said nucleotide sequence is a synthetic DNA sequence that has been designed for expression in a plant.
 12. A vector comprising the DNA construct of claim
 11. 13. A host cell comprising the vector of claim
 12. 14. The host cell of claim 13, wherein the host cell is a plant cell.
 15. A transgenic plant comprising the host cell of claim
 14. 16. A composition comprising the host cell of claim
 9. 17. The composition of claim 16, wherein said composition is selected from the group consisting of a powder, dust, pellet, granule, spray, emulsion, colloid, and solution.
 18. A method for producing a polypeptide with pesticidal activity, comprising culturing the host cell of claim 8 under conditions in which the nucleic acid molecule encoding the polypeptide is expressed.
 19. A plant having stably incorporated into its genome a DNA construct comprising a nucleotide sequence that encodes a polypeptide having pesticidal activity, wherein said polypeptide comprises an amino acid sequence having at least 90% percent sequence identity to an amino acid sequence set forth in SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, wherein said polypeptide has pesticidal activity.
 20. A transgenic seed of the plant of claim
 19. 21. A method for protecting a plant from an insect pest, said method comprising growing a plant or cell thereof that expresses a nucleotide sequence that encodes a pesticidal polypeptide, wherein said pesticidal polypeptide comprises an amino acid sequence having at least 90% percent sequence identity to an amino acid sequence set forth in SEQ ID Nos: selected from the group consisting of sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, wherein said pesticial polypeptide has pesticide activity.
 22. The method of claim 21, wherein said plant or cell thereof produces a pesticidal polypeptide having pesticidal activity against a lepidopteran, a hemipteran, and/or coleopteran pest.
 23. A method for increasing yield in a plant comprising growing in a field a plant or seed thereof having stably incorporated into its genome a DNA construct comprising a promoter that drives expression in a plant operably linked to a nucleotide sequence that encodes a pesticidal polypeptide, wherein said pesticidal polypeptide comprises nucleotide sequence comprises an amino acid sequence having at least 90% percent sequence identity to an amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, and 205, wherein said pesticidal polypeptide has pesticidal activity, and wherein the field is infested with a pest against which said polypeptide has pesticidal activity. 